Diagnosis kit and chip for bladder cancer using bladder cancer specific methylation marker gene

ABSTRACT

The present invention relates to a kit and nucleic acid chip for diagnosing bladder cancer using a bladder cancer-specific marker gene. More particularly, the invention relates to a kit and nucleic acid chip for diagnosing bladder cancer, which can detect the promoter methylation of a bladder cancer-specific gene, the promoter or exon region of which is methylated specifically in transformed cells of bladder cancer. The use of the diagnostic kit or nucleic acid chip of the invention enables diagnosis of bladder cancer at an early stage of transformation, thus enabling early diagnosis of bladder cancer, and can diagnose bladder cancer in a more accurate and rapid manner compared to a conventional method.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation-in-part application under 35 U.S.C. §120 of U.S. patent application Ser. No. 15/016,424 filed on Feb. 5, 2016, which in turn is a continuation-in-part application of U.S. patent application Ser. No. 13/627,474, now U.S. Pat. No. 9,365,900, filed on Sep. 26, 2012, which in turn is a divisional application of U.S. patent application Ser. No. 12/744,491 filed on Jun. 24, 2010, now abandoned, entitled “DIAGNOSIS KIT AND CHIP FOR BLADDER CANCER USING BLADDER CANCER SPECIFIC METHYLATION MARKER GENE” in the name of Sung Wan A N, et al, which is a U.S. national stage application under the provisions of 35 U.S.C. §371 of International Patent Application No. PCT/KR2008/007081 filed on Dec. 1, 2008, which claims priority of Korean Patent Application No. 10-2007-0124015 filed on Nov. 30, 2007, all of which are hereby incorporated by reference herein in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 3, 2017, is named 322DIV2CIP3_SeqID_ST25_REV.txt and is 112,806 bytes in size.

TECHNICAL FIELD

The present invention relates to a kit and nucleic acid chip for diagnosing bladder cancer using a bladder cancer-specific marker gene, and more particularly to a kit and nucleic acid chip for diagnosing bladder cancer, which can detect the promoter methylation of a bladder cancer-specific gene, the promoter region of which is methylated specifically in transformed cells of bladder cancer.

BACKGROUND ART

Bladder cancer is the most frequent cancer of the urinary system and was found to be caused by many factors. It is known that bladder cancer is mainly caused by smoking or various chemical substances (paints for leather, air pollutants, artificial sweetening agents, nitrates and the like) which irritate the bladder wall while they are excreted as urine after being absorbed in vivo.

As conventional methods for diagnosing bladder cancer, a method of finding abnormal cells in urine is used, but has low accuracy. Also, cystoscopy comprising inserting a catheter into the bladder and collecting suspected tissue from the bladder is an invasive method having relatively high accuracy.

Generally, when bladder cancer is diagnosed at an early stage, the survival rate of bladder cancer patients is increased, but it is not easy to diagnose bladder cancer at an early stage. As a method for diagnosing bladder cancer, a method of incising part of the body is currently being used, but it has difficulty in diagnosing bladder cancer at an early stage.

Bladder cancers are classified, according to invasion into the muscular layer of the bladder, into superficial cancer and invasive cancer. Generally, about 30% of patients upon diagnosis of bladder cancer are invasive bladder cancer patients. Thus, in order to increase the survival period of patients, it is the best method to diagnose bladder cancer at early stage when the bladder cancer lesions are small. Accordingly, there is an urgent need to development a diagnostic method more efficient than various prior diagnostic methods for bladder cancer, that is, a bladder cancer-specific biomarker which allows early diagnosis of bladder cancer, can treat a large amount of samples and has high sensitivity and specificity.

Recently, methods of diagnosing cancer through the measurement of DNA methylation have been suggested. DNA methylation occurs mainly on the cytosine of CpG islands in the promoter region of a specific gene to interfere with the binding of transcription factors, thus silencing the expression of the gene. Thus, detecting the methylation of CpG islands in the promoter of tumor inhibitory genes greatly assists in cancer research. Recently, an attempt has been actively made to determine promoter methylation, by methods such as methylation-specific PCR (hereinafter referred to as MSP) or automatic DNA sequencing, for the diagnosis and screening of cancer.

Although there are disputes on whether the methylation of promoter CpG islands directly induces cancer development or causes a secondary change after cancer development, it has been found that tumor suppressor genes, DNA repair genes, cell cycle regulatory genes and the line in several cancers are hyper-methylated, and thus the expression of these genes are silenced. Particularly, it is known that the hyper-methylation of the promoter region of a specific gene occurs at an early stage of cancer development.

Thus, the methylation of the promoter methylation of tumor-associated genes is an important indication of cancer and can be used in many applications, including the diagnosis and early diagnosis of cancer, the prediction of cancer development, the prediction of prognosis of cancer, follow-up examination after treatment, and the prediction of responses to anticancer therapy. Recently, an actual attempt to examine the promoter methylation of tumor-associated genes in blood, sputum, saliva, feces and to use the examined results for diagnosis and treatment of various cancers has been actively made (Esteller, M. et al., Cancer Res., 59:67, 1999; Sanchez-Cespedez, M. et al., Cancer Res., 60:892, 2000; Ahlquist, D. A. et al., Gastroenterol., 119:1219, 2000).

Accordingly, the present inventors have made many efforts to develop a diagnostic kit capable of effectively diagnosing bladder cancer and, as a result, have found that bladder cancer can be diagnosed by measuring the methylation degree using as a biomarker the promoter of methylation-associated genes which are expressed specifically in bladder cancer cells, thereby completing the present invention.

SUMMARY OF INVENTION

It is, therefore, an object of the present invention to provide a kit for diagnosing bladder cancer, which comprises the methylated promoter or exon region of a bladder cancer marker gene.

Another object of the present invention is to provide a nucleic acid chip for diagnosing bladder cancer, which comprises a probe capable of hybridizing with a fragment containing the CpG island of the bladder cancer-specific marker gene.

Still another object of the present invention is to provide a method for measuring the methylation of the promoter or exon region of a gene originated from a clinical sample.

To achieve the above objects, the present invention provides a kit for diagnosing bladder cancer, which comprises the methylated promoter or exon region of a bladder cancer marker gene selected from the group consisting of: (1) CDX2 (NM_001265)—caudal type homeobox transcription factor 2; (2) CYP1B1 (NM_000104)—cytochrome P450, family 1, subfamily B, polypeptide 1; (3) VSX1 (NM_199425)—visual system homeobox 1 homolog, CHX10-like (zebrafish); (4) HOXA11 (NM_005523)—homeobox A11; (5) T (NM_003181)—T, brachyury homolog (mouse); (6) TBX5 (NM_080717)—T-box 5; (7) PENK (NM_006211)—proenkephalin; (8) PAQR9 (NM_198504)—progestin and adipoQ receptor family member IV; (9) LHX2 (NM_004789)—LIM Homeobox 2; and (10) SIM2 (U80456)—single-minded homog 2 (Drosophila).

The present invention also provides a nucleic acid chip for diagnosing bladder cancer, which comprises a probe capable of hybridizing with a fragment containing the CpG island of the promoter or exon region of the bladder cancer marker gene selected from the group consisting of: (1) CDX2 (NM_001265)—caudal type homeobox transcription factor 2; (2) CYP1B1 (NM_000104)—cytochrome P450, family 1, subfamily B, polypeptide 1; (3) VSX1 (NM_199425)—visual system homeobox 1 homolog, CHX10-like (zebrafish); (4) HOXA11 (NM_005523)—homeobox A11; (5) T (NM_003181)—T, brachyury homolog (mouse); (6) TBX5 (NM_080717)—T-box 5; (7) PENK (NM_006211)—proenkephalin; (8) PAQR9 (NM_198504)—progestin and adipoQ receptor family member IV; (9) LHX2 (NM_004789)—LIM Homeobox 2; and (10) SIM2 (U80456)—single-minded homog 2 (Drosophila).

The present invention also provides a method for detecting the methylation of the promoter or exon region of a clinical sample-originated gene selected from the group consisting of CDX2, CYP1B1, VSX1, HOXA11, T, TBX5, PENK, PAQR9, LHX2 and SIM2.

Other features and embodiments of the present invention will be more apparent from the following detailed description and the appended claims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram showing a process of discovering a methylated biomarker for diagnosis of bladder cancer from the urinary cells of normal persons and bladder cancer patients through CpG micrroarray analysis.

FIG. 2 quantitatively shows the methylation degree obtained through pyrosequencing of 10 methylation biomarkers in bladder cancer cell lines.

FIG. 3A shows measurement results for the methylation indexes of the CDX2, the CYP1B1 and the T biomarker genes in clinical samples. FIG. 3A shows measurement results for the methylation degrees of the CDX2, the CYP1B1 and the T biomarker genes in the urinary cells of normal persons, Cystitis patients, hematuria patients and bladder cancer patients.

FIG. 3B shows measurement results for the methylation indexes of the TBX5, the LHX2 and the SIM2 biomarker genes in clinical samples. FIG. 3B shows measurement results for the methylation degrees of the TBX5, the LHX2 and the SIM2 biomarker genes in the urinary cells of normal persons, Cystitis patients, hematuria patients and bladder cancer patients.

FIG. 3C shows measurement results for the methylation indexes of the VSX1, the HOXA11 and the PENK biomarker genes in clinical samples. FIG. 3C shows measurement results for the methylation degrees of the VSX1, the HOXA11 and the PENK biomarker genes in the urinary cells of normal persons, Cystitis patients, hematuria patients and bladder cancer patients.

FIG. 3D shows measurement results for the methylation indexes of the PAQR9 biomarker genes in clinical samples. FIG. 3D shows measurement results for the methylation degrees of the PAQR9 biomarker genes in the urinary cells of normal persons, Cystitis patients, hematuria patients and bladder cancer patients.

FIG. 4A shows the results of receiver operating characteristic (ROC) curve analysis conducted to measure the sensitivity and specificity of the CDX2 and the CYP1B1 methylation biomarkers for diagnosis of bladder cancer.

FIG. 4B shows the results of receiver operation characteristic (ROC) curve analysis conducted to measure the sensitivity and specificity of the VSX1 and the HOXA11 methylation biomarkers for diagnosis of bladder cancer.

FIG. 4C shows the results of receiver operating characteristic (ROC) curve analysis conducted to measure the sensitivity and specificity of the T and the TBX5 methylation biomarkers for diagnosis of bladder cancer.

FIG. 4D shows the results of receiver operating characteristic (ROC) curve analysis conducted to measure the sensitivity and specificity of the PENK and the PAQR9 methylation biomarkers for diagnosis of bladder cancer.

FIG. 4E shows the results of receiver operating characteristic (ROC) curve analysis conducted to measure the sensitivity and specificity of the LHX2 and the SIM2 methylation biomarkers for diagnosis of bladder cancer.

FIG. 5 shows the frequency of methylation in the urinary cells of normal persons and bladder cancer patients.

FIGS. 6A-6D shows the methylation profile of an optimal panel of 6 biomarker genes for bladder cancer diagnosis (FIG. 6A), selected from among 10 biomarkers using logistic regression analysis, and shows the sensitivity and specificity of the gene panel for diagnosis of bladder cancer (FIG. 6B-D).

FIG. 7 shows the results of PCR performed using the methylated DNA-specific binding protein MBD in order to measure the methylation of the biomarker SIM2 gene for bladder cancer cell in bladder cancer cell lines.

DETAILED DESCRIPTION OF THE INVENTION, AND PREFERRED EMBODIMENTS

In one aspect, the present invention relates to a kit for diagnosing bladder cancer, which comprises the methylated promoter or exon region of a bladder cancer marker gene.

In another aspect, the present invention relates to a nucleic acid chip for diagnosing bladder cancer, which comprises a probe capable of hybridizing with a fragment containing the CpG island of the promoter or exon region of a bladder cancer marker gene.

In the present invention, the promoter or exon region may contain at least one methylated CpG dinucleotide. Also, the promoter or exon region is any one of DNA sequences represented in SEQ ID NO: 31 to SEQ ID NO: 40.

In the present invention, the probe preferably has a size ranging from 10 bp to 1 kb, and has a homology with a base sequence containing the CpG island of the promoter or exon region of a bladder cancer marker gene, such that it can hybridize with the base sequence. More preferably, the probe has a size of 10-100 bp, and has a homology with a base sequence containing the CpG island of the promoter or exon region of a bladder cancer marker gene, such that it can hybridize with the base sequence in strict conditions. If the size of the probe is less than 10 bp, non-specific hybridization will occur, and if it is more than 1 kb, the binding between the probes will occur, thus making it difficult to read hybridization results.

A method for screening a methylation marker gene according to the present invention comprises the steps of: (a) isolating genomic DNAs from transformed cells and non-transformed cells; (b) reacting the isolated genomic DNAs to with a protein binding to methylated DNA and isolating methylated DNAs from the genomic DNAs; and (c) amplifying the isolated methylated DNAs, hybridizing the amplified DNAs to CpG microarrays, and selecting a methylation marker gene showing the greatest difference in methylation degree between normal cells and cancer cells among from the hybridized genes.

By the method for screening the methylation biomarker gene, it is possible to screen various genes, which are methylated not only in bladder cancer, but also in various dysplasic stages which progress to bladder cancer. The screened genes are also useful for blood cancer screening, risk assessment, prognosis, disease identification, disease staging, and selection of therapeutic targets.

The identification of the methylated gene in bladder cancer and abnormalities at various stages enables early diagnosis of bladder cancer in an accurate and effective manner, and allows establishment of methylation data using multiple genes and identification of new therapeutic targets. Additionally, methylation data according to the present invention enables establishment of a more accurate system for diagnosing bladder cancer, when it is used together with a method for detecting other non-methylation-associated biomarkers.

The inventive method enables diagnosis of bladder cancer progression at various stages by determining the methylation stage of at least one nucleic acid biomarker obtained from a sample. When the methylation stage of nucleic acid isolated from a sample at each stage of bladder cancer is compared with the methylation stage of at least one nucleic acid obtained from a sample having no abnormality in the cell proliferation of bladder tissue, a certain stage of bladder cancer in the sample can be determined. The methylation stage may be hypermethylation.

In one embodiment of the present invention, nucleic acid can be methylated in the regulatory region of a gene. In another embodiment, since methylation begins from the outer boundary of the regulatory region of a gene and then spreads inward, detection of methylation at the outer boundary of the regulatory region enables early diagnosis of genes which are involved in cell transformation.

In still another embodiment of the present invention, the cell growth abnormality (dysplasia) of bladder tissue can be diagnosed by detecting the methylation of at least one nucleic acid of the following nucleic acids using a kit or a nucleic acid chip: CDX2 (NM_001265, caudal type homeobox transcription factor 2); CYP1B1 (NM_000104, cytochrome P450, family 1, subfamily B, polypeptide 1); VSX1 (NM_199425, visual system homeobox 1 homolog, CHX10-like (zebrafish)); HOXA11 (NM_005523, homeobox A11); T (NM_003181, T, brachyury homolog (mouse)); TBX5 (NM_080717, T-box 5); PENK (NM_006211, proenkephalin); and PAQR9 (NM_198504, progestin and adipoQ receptor family member IV); LHX2 (NM_004789) LIM Homeobox 2; SIM2 (U80456), single-minded homog 2 (Drosophila) gene and combination thereof.

The use of the diagnostic kit or nucleic acid chip of the present invention can determine the cell growth abnormality of bladder tissue in a sample. The method for determining the cell growth abnormality of bladder tissue comprises determining the methylation of at least one nucleic acid isolated from a sample. In the method, the methylation stage of at least one nucleic acid is compared with the methylation stage of a nucleic acid isolated from a sample having no cell growth abnormality (dysplasia).

The examples of said nucleic acid are follows: CDX2 (NM_001265, caudal type homeobox transcription factor 2); CYP1B1 (NM_000104, cytochrome P450, family 1, subfamily B, polypeptide 1); VSX1 (NM_199425, visual system homeobox 1 homolog, CHX10-like (zebrafish)); HOXA11 (NM_005523, homeobox A11); T (NM_003181, T, brachyury homolog (mouse)); TBX5 (NM_080717, T-box 5); PENK (NM_006211, proenkephalin); and PAQR9 (NM_198504, progestin and adipoQ receptor family member IV); LHX2 (NM_004789) LIM Homeobox 2; SIM2 (U80456), single-minded homog 2 (Drosophila) gene and combination thereof.

In still another embodiment of the present invention, cells capable of forming bladder cancer can be diagnosed at an early stage using the methylation gene marker. When genes confirmed to be methylated in cancer cells are methylated in cells which seem to be normal clinically or morphologically, the cells that seem to be normal are cells, the carcinogenesis of which is in progress. Thus, bladder cancer can be diagnosed at an early stage by detecting the methylation of bladder cancer-specific genes in the cells that seem to be normal.

The use of the methylation marker gene of the present invention enables detection of the cell growth abnormality (dysplasia progression) of bladder tissue in a sample. The method for detecting the cell growth abnormality (dysplasia progression) of bladder tissue comprises bringing at least one nucleic acid isolated from a sample into contact with an agent capable of determining the methylation status of the nucleic acid. The method comprises determining the methylation status of at least one region in at least one nucleic acid, and the methylation status of the nucleic acid differs from the methylation status of the same region in a nucleic acid isolated from a sample having no cell growth abnormality (dysplasia progression) of bladder tissue.

In still another embodiment of the present invention, transformed bladder cancer cells can be detected by examining the methylation of a marker gene using the above-described kit or nucleic acid chip.

In still another embodiment of the present invention, bladder cancer can be diagnosed by examining the methylation of a marker gene using the above-described kit or nucleic acid chip.

In still another embodiment of the present invention, the likelihood of progression to bladder cancer can be diagnosed by examining the methylation of a marker gene with the above-described kit or nucleic acid chip in a sample showing a normal phenotype. The sample may be solid or liquid tissue, cell, urine, serum or plasma.

In still another aspect, the present invention relates to a method for detecting the promoter methylation of a clinical sample-originated gene.

In the present invention, the method for measuring the promoter methylation of a clinical sample-originated gene may be selected from the group consisting of PCR, methylation specific PCR, real-time methylation specific PCR, PCR using a methylated DNA-specific binding protein, quantitative PCR, pyrosequencing and bisulfite sequencing, and the clinical sample is preferably a tissue, cell, blood or urine originated from patients suspected of cancer or subjects to be diagnosed.

In the present invention, the method for detecting the promoter methylation of the gene comprises the steps of: (a) isolating a sample DNA from a clinical sample; (b) amplifying the isolated DNA with primers capable of amplifying a fragment containing the promoter CpG island of a gene selected from the group consisting of CDX2, CYP1B1, VSX1, HOXA11, T, TBX5, PENK, PAQR9, LHX2 and SIM2; and (c) determining the promoter methylation of the DNA on the basis of whether the DNA has been amplified or not in step (b).

In an embodiment of the present disclosure, primer(s) that could amplify a methylated CpG of SIM2 might be used, and such primer(s) comprises at least one or more CpG dinucleotide in a region which hybridizes to the methylated CpG of SIM2 Specifically, the primer(s) for amplifying a methylated CpG of SIM2 comprise sequence(s) having a homology of 50% or more with sequence(s) selected from the group consisting of SEQ ID NOs: 43-44, 46-63, 65-126, 128-189, 191-232, 234-295, 297-358, 360-421, and 423-460. Preferably, the primer(s) for amplifying a methylated CpG of SIM2 comprise sequence(s) having a homology of at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100% with sequence(s) selected from the group consisting of SEQ ID NOs: 43-44, 46-63, 65-126, 128-189, 191-232, 234-295, 297-358, 360-421, and 423-460.

If required, probe(s) capable of hybridizing with a methylated CpG of SIM2 might be used. The probe(s) capable of hybridizing with a methylated CpG of SIM2 comprise at least one or more CpG dinucleotide in a region which hybridizes to the methylated CpG of SIM2. Specifically, probe(s) might comprise sequence(s) having a homology of 50% or more with sequence(s) selected from the group consisting of SEQ ID NOs: 45, 64, 127, 190, 233, 296, 359, 422 and 461. Preferably, the probe(s) capable of hybridizing with a methylated CpG of SIM2 comprise sequence(s) having a homology of at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100% with sequence(s) selected from the group consisting of SEQ ID NOs: 45, 64, 127, 190, 233, 296, 359, 422 and 461.

In another embodiment of the present invention, the likelihood of development of tissue to bladder cancer can be evaluated by examining the methylation frequency of a gene which is methylated specifically in bladder cancer and determining the methylation frequency of tissue having the likelihood of progression to bladder cancer.

As used herein, “cell conversion” refers to the change in characteristics of a cell from one form to another such as from normal to abnormal, non-tumorous to tumorous, undifferentiated to differentiated, stem cell to non-stem cell. Further, the conversion may be recognized by morphology of the cell, phenotype of the cell, biochemical characteristics and so on.

As used herein, the term “early diagnosis” of cancer refers to discovering the likelihood of cancer before metastasis. Preferably, it refers to discovering the likelihood of cancer before a morphological change in a sample tissue or cell is observed. Additionally, the term “early diagnosis” of transformation the high probability of a cell to undergo transformation in its early stages before the cell is morphologically designated as being transformed.

As used herein, the term “hypermethylation” refers to the methylation of CpG islands.

As used herein, the term “sample” or “biological sample” is referred to in its broadest sense, and includes any biological sample obtained from an individual, body fluid, cell line, tissue culture or other sources, according to the type of analysis that is to be performed. Methods of obtaining body fluid and tissue biopsy from mammals are generally widely known. A preferred source is bladder biopsy.

Screening for Methylation Regulated Biomarkers

The present invention is directed to a method of determining biomarker genes that are methylated when the cell or tissue is converted or changed from one type of cell to another. As used herein, “converted” cell refers to the change in characteristics of a cell or tissue from one form to another such as from normal to abnormal, non-tumorous to tumorous, undifferentiated to differentiated and so on.

In one Example of the present invention, urinary cells were isolated from the urine of normal persons and bladder cancer patients, and then genomic DNAs were isolated from the urinary cells. In order to obtain only methylated DNAs from the genomic DNAs, the genomic DNAs were allowed to react with McrBt binding to methylated DNA, and then methylated DNAs binding to the McrBt protein were isolated. The isolated methylated DNAs binding to the McrBt protein were amplified, and then the DNAs originated from the normal persons were labeled with Cy3, and the DNAs originated from the bladder cancer patients were labeled with Cy5. Then, the DNAs were hybridized to human CpG-island microarrays, and 10 genes showing the greatest difference in methylation degree between the normal persons and the bladder cancer patients were selected as biomarkers.

In the present invention, in order to further confirm whether the 10 biomarkers have been methylated, pyrosequencing was performed.

Specifically, total genomic DNA was isolated from the bladder cell lines RT-4, J82, HT1197 and HT1376 and treated with bisulfite. The genomic DNA converted with bisulfite was amplified. Then, the amplified PCR product was subjected to pyrosequencing in order to measure the methylation degree of the genes. As a result, it could be seen that the 10 biomarkers were all methylated.

Biomarker for Bladder Cancer

The present invention provides a biomarker for diagnosing bladder cancer.

Biomarkers for Bladder Cancer—Using Cancer Cells for Comparison with Normal Cells

In one embodiment of the present invention, it is understood that “normal” cells are those that do not show any abnormal morphological or cytological changes. “Tumor” cells mean cancer cells. “Non-tumor” cells are those cells that were part of the diseased tissue but were not considered to be the tumor portion.

In one aspect, the present invention is based on the relationship between bladder cancer and the hypermethylation of the promoter or exon region of the following 10 genes: CDX2 (NM_001265, caudal type homeobox transcription factor 2); CYP1B1 (NM_000104, cytochrome P450, family 1, subfamily B, polypeptide 1); VSX1 (NM_199425, visual system homeobox 1 homolog, CHX10-like (zebrafish)); HOXA11 (NM_005523, homeobox A11); T (NM_003181, T, brachyury homolog (mouse)); TBX5 (NM_080717, T-box 5); PENK (NM_006211, proenkephalin); and PAQR9 (NM_198504, progestin and adipoQ receptor family member IV); LHX2 (NM_004789)—LIM Homeobox 2; and SIM2 (U80456)—single-minded homolog 2 (Drosophila); gene.

With other applications of the diagnostic kit or nucleic acid chip of the present invention, the invention can diagnose a cellular proliferative disorder of bladder tissue in a subject by determining the state of methylation of one or more nucleic acids isolated from the subject, wherein the state of methylation of one or more nucleic acids as compared with the state of methylation of one or more nucleic acids from a subject not having the cellular proliferative disorder of bladder tissue is indicative of a cellular proliferative disorder of bladder tissue in the subject. A preferred nucleic acid is a CpG-containing nucleic acid, such as a CpG island.

With other applications of the diagnostic kit or nucleic acid chip of the present invention, the cell growth abnormality of bladder tissue in a subject can be diagnosed comprising determining the methylation of one or more nucleic acids isolated from the subject. Said nucleic acid is preferably encoding the followings: CDX2 (NM_001265, caudal type homeobox transcription factor 2); CYP1B1 (NM_000104, cytochrome P450, family 1, subfamily B, polypeptide 1); VSX1 (NM_199425, visual system homeobox 1 homolog, CHX10-like (zebrafish)); HOXA11 (NM_005523, homeobox A11); T (NM_003181, T, brachyury homolog (mouse)); TBX5 (NM_080717, T-box 5); PENK (NM_006211, proenkephalin); and PAQR9 (NM_198504, progestin and adipoQ receptor family member IV); LHX2 (NM_004789)—LIM Homeobox 2; and SIM2 (U80456)—single-minded homolog 2 (Drosophila); gene and combinations thereof. The state of methylation of one or more nucleic acids as compared with the state of methylation of said nucleic acid from a subject not having a predisposition to the cellular proliferative disorder of bladder tissue is indicative of a cell proliferative disorder of bladder tissue in the subject.

As used herein, “predisposition” refers to an increased likelihood that an individual will have a disorder. Although a subject with a predisposition does not yet have the disorder, there exists an increased propensity to the disease.

Another embodiment of the invention provides a method for diagnosing a cellular proliferative disorder of bladder tissue in a subject comprising contacting a nucleic acid-containing specimen from the subject with an agent that provides a determination of the methylation state of nucleic acids in the specimen, and identifying the methylation state of at least one region of at least one nucleic acid, wherein the methylation state of at least one region of at least one nucleic acid that is different from the methylation state of the same region of the same nucleic acid in a subject not having the cellular proliferative disorder is indicative of a cellular proliferative disorder of bladder tissue in the subject.

The inventive method includes determining the state of methylation of one or more regions of one or more nucleic acids isolated from the subject. The phrases “nucleic acid” or “nucleic acid sequence” as used herein refer to an oligonucleotide, nucleotide, polynucleotide, or to a fragment of any of these, to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded, to DNA or RNA of genomic or synthetic origin which may represent a sense or antisense strand, peptide nucleic acid (PNA), or to any DNA-like or RNA-like material of natural or synthetic origin. As will be understood by those of skill in the art, when the nucleic acid is RNA, the deoxynucleotides A, G, C, and T are replaced by ribonucleotides A, G, C, and U, respectively.

The nucleic acid of interest can be any nucleic acid where it is desirable to detect the presence of a differentially methylated CpG island. The CpG island is a CpG rich region of a nucleic acid sequence.

Methylation

Any nucleic acid sample, in purified or nonpurified form, can be utilized in accordance with the present invention, provided it contains or is suspected of containing, a nucleic acid sequence containing a target locus (e.g., CpG-containing nucleic acid). One nucleic acid region capable of being differentially methylated is a CpG island, a sequence of nucleic acid with an increased density relative to other nucleic acid regions of the dinucleotide CpG. The CpG doublet occurs in vertebrate DNA at only about 20% of the frequency that would be expected from the proportion of G*C base pairs. In certain regions, the density of CpG doublets reaches the predicted value; it is increased by ten fold relative to the rest of the genome. CpG islands have an average G*C content of about 60%, and general DNA have an average G*C contents of about 40%. The islands take the form of stretches of DNA typically about one to two kilobases long. There are about 45,000 such islands in the human genome.

In many genes, the CpG islands begin just upstream of a promoter and extend downstream into the transcribed region. Methylation of a CpG island at a promoter usually prevents expression of the gene. The islands can also surround the 5′ region of the coding region of the gene as well as the 3′ region of the coding region. Thus, CpG islands can be found in multiple regions of a nucleic acid sequence including upstream of coding sequences in a regulatory region including a promoter region, in the coding regions (e.g., exons), in downstream of coding regions, for example, enhancer regions, and in introns.

In general, the CpG-containing nucleic acid is DNA. However, invention methods may employ, for example, samples that contain DNA, or DNA and RNA, including messenger RNA, wherein DNA or RNA may be single stranded or double stranded, or a DNA-RNA hybrid may be included in the sample.

A mixture of nucleic acids may also be employed. The specific nucleic acid sequence to be detected may be a fraction of a larger molecule or can be present initially as a discrete molecule, so that the specific sequence constitutes the entire nucleic acid. It is not necessary that the nucleic acid sequence is present initially in a pure form, the nucleic acid may be a minor fraction of a complex mixture, such as contained in whole human DNA. The nucleic acid-containing sample used for determination of the state of methylation of nucleic acids contained in the sample or detection of methylated CpG islands may be extracted by a variety of techniques such as that described by Sambrook, et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989; incorporated in its entirety herein by reference).

A nucleic acid can contain a regulatory region which is a region of DNA that encodes information or controls transcription of the nucleic acid. Regulatory regions include at least one promoter. A “promoter” is a minimal sequence sufficient to direct transcription, to render promoter-dependent gene expression controllable for cell-type specific, tissue-specific, or inducible by external signals or agents. Promoters may be located in the 5′ or 3′ regions of the gene. Promoter regions, in whole or in part, of a number of nucleic acids can be examined for sites of CpG-island methylation. Moreover, it is generally recognized that methylation of the target gene promoter proceeds naturally from the outer boundary inward. Therefore, early stage of cell conversion can be detected by assaying for methylation in these outer areas of the promoter region.

Nucleic acids isolated from a subject are obtained in a biological specimen from the subject. If it is desired to detect bladder cancer or stages of bladder cancer progression, the nucleic acid may be isolated from bladder tissue by scraping or taking a biopsy. These specimens may be obtained by various medical procedures known to those of skill in the art.

In one aspect of the invention, the state of methylation in nucleic acids of the sample obtained from a subject is hypermethylation compared with the same regions of the nucleic acid in a subject not having the cellular proliferative disorder of bladder tissue. Hypermethylation, as used herein, is the presence of methylated alleles in one or more nucleic acids. Nucleic acids from a subject not having a cellular proliferative disorder of bladder tissues contain no detectable methylated alleles when the same nucleic acids are examined.

Sample

The present invention describes early diagnosis of bladder cancer and utilizes the methylation of bladder cancer-specific genes. The methylation of bladder cancer-specific genes also occurred in tissue near tumor sites. Therefore, in the method for early diagnosis of bladder cancer, the methylation of bladder cancer-specific genes can be detected by examining all samples including liquid or solid tissue. The samples include, but are not limited to, tissue, cell, urine, serum or plasma.

Individual Genes and Panel

It is understood that the present invention may be practiced using each gene separately as a diagnostic or prognostic marker, or a few marker genes combined into a panel display format so that several marker genes may be detected to increase reliability and efficiency. Further, any of the genes identified in the present application may be used individually or as a set of genes in any combination with any of the other genes that are recited in the application. Also, genes may be ranked and weighted according to their importance together with the number of genes that are methylated, and a level of likelihood of development to cancer can be assigned. Such algorithms are within the scope of the present invention.

Methylation Detection Methods Methylation Specific PCR

When genomic DNA is treated with bisulfite, the methylated cytosine in the 5′-CpG′-3 region remains without changes, and unmethylated cytosine is changed to uracil. Thus, for a base sequence modified by bisulfite treatment, PCR primers corresponding to regions in which a 5′-CpG-3′ base sequence is present were constructed. Herein, two kinds of primers corresponding to the methylated case and the unmethylated case were constructed. When genomic DNA is modified with bisulfite and then subjected to PCR using the two kinds of primers, in the case in which the DNA is methylated, a PCR product is made from the DNA in which the primers corresponding to the methylated base sequence are used. In contrast, in the case in which the gene is unmethylated, a PCR product is made from the DNA in which the primers corresponding to the unmethylated base sequence are used. The methylation of DNA can be qualitatively analyzed using agarose gel electrophoresis.

Real-Time Methylation-Specific PCR

Real-time methylation-specific PCR is a real-time measurement method modified from methylation-specific PCR, and comprises treating genomic DNA with bisulfite, designing PCR primers corresponding to the methylated case and performing real-time PCR using the primers. Herein, methods of detecting methylation include two methods: a method of performing detection using a TanMan probe complementary to the amplified base sequence, and a method of performing detection using Sybergreen. Thus, real-time methylation-specific PCR selectively quantitatively analyze only DNA. Herein, a standard curve was prepared using an in vitro methylated DNA sample, and for standardization, a gene having no 5′-CpG-3′ sequence in the base sequence was also amplified as a negative control group and was quantitatively analyzed for the methylation degree.

Pyrosequencing

Pyrosequencing is a real-time sequencing method modified from a bisulfite sequencing method. In the same manner as bisulfite sequencing, genomic DNA was modified by bisulfite treatment, and then primers corresponding to a region having no 5′-CpG-3′ base sequence were constructed. After the genomic DNA had been treated with bisulfite, it was amplified with the PCR primers, and then subjected to real-time sequence analysis using sequencing primers. The amounts of cytosine and thymine in the 5′-CpG-3′ region were quantitatively analyzed, and the methylation degree was expressed as a methylation index.

PCR or Quantitative PCR Using Methylated DNA-Specific Binding Protein and DNA Chip

In a PCR or DNA chip method using a methylated DNA-specific binding protein, when a protein binding specifically only to methylated DNA is mixed with DNA, the protein binds specifically only to methylated DNA, and thus only methylated DNA can be isolated. In the present invention, genomic DNA was mixed with a methylated DNA-specific binding protein, and then only methylated DNA was selectively isolated. The isolated DNA was amplified using PCR primers corresponding to the promoter region thereof, and then the methylation of the DNA was measured by agarose gel electrophoresis.

In addition, the methylation of DNA can also be measured by a quantitative PCR method. Specifically, methylated DNA isolated using a methylated DNA-specific binding protein can be labeled with a fluorescent dye and hybridized to a DNA chip in which complementary probes are integrated, thus measuring the methylation of the DNA. Herein, the methylated DNA-specific binding protein is not limited to McrBt.

Detection of Differential Methylation-Methylation Sensitive Restriction Endonuclease

Detection of differential methylation can be accomplished by contacting a nucleic acid sample with a methylation sensitive restriction endonuclease that cleaves only unmethylated CpG sites under conditions and for a time to allow cleavage of unmethylated nucleic acid.

In a separate reaction, the sample is further contacted with an isoschizomer of the methylation sensitive restriction endonuclease that cleaves both methylated and unmethylated CpG-sites under conditions and for a time to allow cleavage of methylated nucleic acid. Specific primers are added to the nucleic acid sample under conditions and for a time to allow nucleic acid amplification to occur by conventional methods. The presence of amplified product in the sample digested with methylation sensitive restriction endonuclease but absence of an amplified product in sample digested with an isoschizomer of the methylation sensitive restriction enzyme endonuclease that cleaves both methylated and unmethylated CpG-sites indicates that methylation has occurred at the nucleic acid region being assayed. However, lack of amplified product in the sample digested with methylation sensitive restriction endonuclease together with lack of an amplified product in the sample digested with an isoschizomer of the methylation sensitive restriction enzyme endonuclease that cleaves both methylated and unmethylated CpG-sites indicates that methylation has not occurred at the nucleic acid region being assayed.

As used herein, a “methylation sensitive restriction endonuclease” is a restriction endonuclease that includes CG as part of its recognition site and has altered activity when the C is methylated as compared to when the C is not methylated (e.g., Sma I). Non-limiting examples of methylation sensitive restriction endonucleases include MspI, HpaII, BssHII, BstUI and Nod. Such enzymes can be used alone or in combination. Other methylation sensitive restriction endonucleases such as SacII and EagI may be applied to the present invention, but are not limited to these enzymes.

An “isoschizomer” of a methylation sensitive restriction endonuclease is a restriction endonuclease that recognizes the same recognition site as a methylation sensitive restriction endonuclease but cleaves both methylated CGs and unmethylated CGs, such as for example, MspI.

Primers of the invention are designed to be “substantially” complementary to each strand of the locus to be amplified and include the appropriate G or C nucleotides as discussed above. This means that the primers must be sufficiently complementary to hybridize with their respective strands under conditions that allow the agent for polymerization to perform. Primers of the invention are employed in the amplification process, which is an enzymatic chain reaction that produces exponentially increasing quantities of target locus relative to the number of reaction steps involved (e.g., polymerase chain reaction (PCR)). Typically, one primer is complementary to the negative (−) strand of the locus (antisense primer) and the other is complementary to the positive (+) strand (sense primer). Annealing the primers to denatured nucleic acid followed by extension with an enzyme, such as the large fragment of DNA Polymerase I (Klenow) and nucleotides, results in newly synthesized+ and − strands containing the target locus sequence. Because these newly synthesized sequences are also templates, repeated cycles of denaturing, primer annealing, and extension results in exponential production of the region (i.e., the target locus sequence) defined by the primer. The product of the chain reaction is a discrete nucleic acid duplex with termini corresponding to the ends of the specific primers employed.

Preferably, the method of amplifying is by PCR, as described herein and as is commonly used by those of ordinary skill in the art. However, alternative methods of amplification have been described and can also be employed such as real time PCR or linear amplification using isothermal enzyme. Multiplex amplification reactions may also be used.

Detection of Differential Methylation-Bifulfite Sequencing Method

Another method for detecting a methylated CpG-containing nucleic acid includes contacting a nucleic acid-containing specimen with an agent that modifies unmethylated cytosine, amplifying the CpG-containing nucleic acid in the specimen by means of CpG-specific oligonucleotide primers, wherein the oligonucleotide primers distinguish between modified methylated and non-methylated nucleic acid and detecting the methylated nucleic acid. The amplification step is optional and although desirable, is not essential. The method relies on the PCR reaction itself to distinguish between modified (e.g., chemically modified) methylated and unmethylated DNA. Such methods are described in U.S. Pat. No. 5,786,146, the contents of which are incorporated herein in their entirety especially as they relate to the bisulfite sequencing method for detection of methylated nucleic acid.

Substrates

Once the target nucleic acid region is amplified, the nucleic acid can be hybridized to a known gene probe immobilized on a solid support to detect the presence of the nucleic acid sequence.

As used herein, “substrate,” when used in reference to a substance, structure, surface or material, means a composition comprising a nonbiological, synthetic, nonliving, planar, spherical or flat surface that is not heretofore known to comprise a specific binding, hybridization or catalytic recognition site or a plurality of different recognition sites or a number of different recognition sites which exceeds the number of different molecular species comprising the surface, structure or material. The substrate may include, for example and without limitation, semiconductors, synthetic (organic) metals, synthetic semiconductors, insulators and dopants; metals, alloys, elements, compounds and minerals; synthetic, cleaved, etched, lithographed, printed, machined and microfabricated slides, devices, structures and surfaces; industrial polymers, plastics, membranes; silicon, silicates, glass, metals and ceramics; wood, paper, cardboard, cotton, wool, cloth, woven and nonwoven fibers, materials and fabrics.

Several types of membranes are known to one of skill in the art for adhesion of nucleic acid sequences. Specific non-limiting examples of these membranes include nitrocellulose or other membranes used for detection of gene expression such as polyvinylchloride, diazotized paper and other commercially available membranes such as GENESCREEN™, ZETAPROBE™ (Biorad), and NYTRAN™. Beads, glass, wafer and metal substrates are included. Methods for attaching nucleic acids to these objects are well known to one of skill in the art. Alternatively, screening can be done in liquid phase.

Hybridization Conditions

In nucleic acid hybridization reactions, the conditions used to achieve a particular level of stringency will vary, depending on the nature of the nucleic acids being hybridized. For example, the length, degree of homology, nucleotide sequence composition (e.g., GC/AT content), and nucleic acid type (e.g., RNA, DNA) of the hybridizing regions of the nucleic acids can be considered in selecting hybridization conditions. An additional consideration is whether one of the nucleic acids is immobilized, for example, on a filter.

An example of progressively higher stringency conditions is as follows: 2×SSC/0.1% SDS at about room temperature (hybridization conditions); 0.2×SSC/0.1% SDS at about room temperature (low stringency conditions); 0.2×SSC/0.1% SDS at about 42° C. (moderate stringency conditions); and 0.1×SSC at about 68° C. (high stringency conditions). Washing can be carried out using only one of these conditions, e.g., high stringency conditions, or each of the conditions can be used, e.g., for 10-15 minutes each, in the order listed above, repeating any or all of the steps listed. However, as mentioned above, optimal conditions will vary, depending on the particular hybridization reaction involved, and can be determined empirically. In general, conditions of high stringency are used for the hybridization of the probe of interest.

Label

The probe of interest can be detectably labeled, for example, with a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator, or an enzyme. Those of ordinary skill in the art will know of other suitable labels for binding to the probe, or will be able to ascertain such, using routine experimentation.

Kit

In accordance with the present invention, there is provided a kit useful for the detection of a cellular proliferative disorder in a subject. Kits according to the present invention include a carrier means compartmentalized to receive a sample therein, one or more containers comprising a first container containing a reagent which sensitively cleaves unmethylated cytosine, a second container containing primers for amplification of a CpG-containing nucleic acid, and a third container containing a means to detect the presence of cleaved or uncleaved nucleic acid. Primers contemplated for use in accordance with the invention include those set forth in SEQ ID NOS: 1-20, and any functional combination and fragments thereof.

In an embodiment of the present disclosure, primer(s) that could amplify a methylated CpG of SIM2 might be used, and such primer(s) comprises at least one or more CpG dinucleotide in a region which hybridizes to the methylated CpG of SIM2 Specifically, the primer(s) for amplifying a methylated CpG of SIM2 comprise sequence(s) having a homology of 50% or more with sequence(s) selected from the group consisting of SEQ ID NOs: 43-44, 46-63, 65-126, 128-189, 191-232, 234-295, 297-358, 360-421, and 423-460. Preferably, the primer(s) for amplifying a methylated CpG of SIM2 comprise sequence(s) having a homology of at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100% with sequence(s) selected from the group consisting of SEQ ID NOs: 43-44, 46-63, 65-126, 128-189, 191-232, 234-295, 297-358, 360-421, and 423-460.

If required, probe(s) capable of hybridizing with a methylated CpG of SIM2 might be used. The probe(s) capable of hybridizing with a methylated CpG of SIM2 comprise at least one or more CpG dinucleotide in a region which hybridizes to the methylated CpG of SIM2. Specifically, probe(s) might comprise sequence(s) having a homology of 50% or more with sequence(s) selected from the group consisting of SEQ ID NOs: 45, 64, 127, 190, 233, 296, 359, 422 and 461. Preferably, the probe(s) capable of hybridizing with a methylated CpG of SIM2 comprise sequence(s) having a homology of at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100% with sequence(s) selected from the group consisting of SEQ ID NOs: 45, 64, 127, 190, 233, 296, 359, 422 and 461.

Functional combination or fragment refers to its ability to be used as a primer to detect whether methylation has occurred on the region of the genome sought to be detected.

Carrier means are suited for containing one or more container means such as vials, tubes, and the like, each of the container means comprising one of the separate elements to be used in the method. In view of the description provided herein of invention methods, those of skill in the art can readily determine the apportionment of the necessary reagents among the container means. For example, one of the container means can comprise a container containing methylation sensitive restriction endonuclease. One or more container means can also be included comprising a primer complementary to the nucleic acid locus of interest. In addition, one or more container means can also be included containing an isoschizomer of the methylation sensitive restriction enzyme.

EXAMPLES

Hereinafter, the present invention will be described in further detail with reference to examples. It is to be understood, however, that these examples are for illustrative purposes only and are not to be construed to limit the scope of the present invention.

Example 1: Discovery of Bladder Cancer-Specific Methylated Genes

In order to screen biomarkers which are methylated specifically in bladder cancer, about 20 ml of the urine of each of 10 bladder cancer patients and 10 normal persons was centrifuged in a centrifuge (Hanil Science Industrial Co., Ltd., Korea) at 4,200×g for 10 minutes to isolate urinary cells. The supernatant was discarded, and the cell precipitate was washed twice with 5 ml of PBS. Genomic DNA was isolated from the cell precipitate using the QIAamp DNA Mini kit (QIAGEN, USA). 500 ng of the isolated genomic DNA was sonicated (Vibra Cell, SONICS), thus constructing about 200-300-bp-genomic DNA fragments.

To obtain only methylated DNA from the genomic DNA, a methyl binding domain (MBD) known to bind to methylated DNA (Fraga et al., Nucleic Acid Res., 31:1765-1774, 2003) was used. Specifically, 2 μg of 6×His-tagged MBD was pre-incubated with 500 ng of the genomic DNA of E. coli JM110 (No. 2638, Biological Resource Center, Korea Research Institute of Bioscience & Biotechnology), and then bound to Ni-NTA magnetic beads (Qiagen, USA). 500 ng of the sonicated genomic DNA isolated from the urinary cells of the normal persons and the bladder cancer patients was allowed to react with the beads in the presence of binding buffer solution (10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 3 mM MgCl₂, 0.1% Triton-X100, 5% glycerol, 25 mg/ml BSA) at 4° C. for 20 minutes. Then, the beads were washed three times with 500 μl of a binding buffer solution containing 700 mM NaCl, and then methylated DNA bound to the MBD was isolated using the QiaQuick PCR purification kit (QIAGEN, USA).

Then, the methylated DNAs bound to the MBD were amplified using a genomic DNA amplification kit (Sigma, USA, Cat. No. WGA2), and 4 μg of the amplified DNAs were labeled with Cy3 for the normal person-originated DNA and with Cy5 for the bladder cancer patient-originated DNA using the BioPrime Total Genomic Labeling system I (Invitrogen Corp., USA). The DNA of the normal persons and the DNA of the bladder patients were mixed with each other, and then hybridized to 244K human CpG microarrays (Agilent, USA) (FIG. 1). After the hybridization, the DNA mixture was subjected to a series of washing processes, and then scanned using an Agilent scanner. The calculation of signal values from the microarray images was performed by calculating the relative difference in signal strength between the normal person sample and the bladder cancer patient sample using Feature Extraction program v. 9.5.3.1 (Agilent).

In order to select unmethylated spots from the normal sample, the whole Cy3 signal values were averaged, and then spots having a signal value of less than 10% of the averaged value were regarded as those unmethylated in the samples of the normal persons. As a result, 41,674 spots having a Cy3 signal value of less than 65 were selected.

In order to select the methylated spots in the samples of the bladder cancer patients from among the 41,674 spots, spots having a Cy5 signal value of more than 130 were regarded as the methylated spots in bladder cancer. As a result, 631 spots having a Cy5 signal value of more than 130 were selected. From these spots, 227 genes corresponding to the promoter region were secured as bladder cancer-specific methylated genes.

From the genes, 10 genes (CDX2, CYP1B1, VSX16, HOXA11, T, TBX5, PENK, PAQR9, LHX2, and SIM2) showing the greatest relative difference between methylation degree of the normal persons and that of the bladder cancer patients were selected, and the presence of CpG islands in the promoter region of the 10 genes was confirmed using MethPrimer. The 10 genes were secured as methylation biomarkers for diagnosis of bladder cancer. The list of the 10 genes and the relative methylation degree thereof in the urinary cells of the bladder patients relative to those of the normal persons are shown in Table 1 below.

TABLE 1 10 methylation biomarkers for diagnosis of bladder cancer Biomarker for Relative bladder cancer GenBank No. Description methylation ^(a) CDX2 NM_001265 caudal type homeobox transcription factor 2 11.0 CYP1B1 NM_000104 cytochrome P450, family 1, subfamily B, 14.6 polypeptide 1 VSX1 NM_199425 visual system homeobox 1 homolog, CHX10-like 33.4 (zebrafish) HOXA11 NM_005523 homeobox A11 14.2 T NM_003181 T, brachyury homolog (mouse) 51.4 TBX5 NM_080717 T-box 5 18.7 PENK NM_006211 Proenkephalin 12.7 PAQR9 NM_198504 progestin and adipoQ receptor family member IX 4.1 LHX2 NM_004789 LIM Homeobox 2 5.8 SIM2 U80456 Single-minded homolog 2 (Drosophila) 9.5 ^(a) Relative methylation degree between the normal sample and the bladder patient sample, calculated by dividing the average signal (Cy5) value in the bladder cancer patient sample in CpG microarrays by the average signal (Cy5) value in the normal person sample.

Example 2: Measurement of Methylation of Biomarker Genes in Cancer Cell Lines

In order to further determine the methylation status of the 10 genes, bisulfite sequencing for each promoter was performed.

In order to modify unmethylated cytosine to uracil using bisulfite, total genomic DNA was isolated from the bladder cancer cell lines RT-4 (Korean Cell Line Bank (KCLB 30002), J82 (KCLB 30001), HT1197 (KCLB 21473) and HT1376 (KCLB 21472), and 200 ng of the genomic DNA was treated with bisulfite using the EZ DNA methylation-gold kit (Zymo Research, USA). When DNA is treated with bisulfite, unmethylated cytosine is modified to uracil, and the methylated cytosine remains without changes. The DNA treated with bisulfite was eluted in 20 μl of sterile distilled water and subjected to pyrosequencing.

PCR and sequencing primers for performing pyrosequencing for the 10 genes were designed using the PSQ assay design program (Biotage, USA). The PCR and sequencing primers for measuring the methylation of each gene are shown in Tables 2 and 3 below.

TABLE 2 Primers and conditions SEQ ID CpG Amplicon Gene Primer Sequence (5′→3′) NO: position^(a) size CDX2 forward TGGTGTTTGTGTTATTATTAATAG  1 -138, -129, 129 bp reverse Biotin-CACCTCCTTCCCACTAAACTA  2 -121, -118 CYP1B1 forward GTAAGGGTATGGGAATTGA  3 +73, +83  90 bp reverse Biotin-CCCTTAAAAACCTAACAAAATC  4 +105 VSX1 forward GGAGTGGGATTGAGGAGATTT  5 -1121, -1114,  89 bp reverse Biotin-AAACCCAACCAACCCTCAT  6 -1104, 1100 HOXA11 forward AGTAAGTTTATGGGAGGGGGATT  7 -415, -405, 243 bp reverse Biotin-  8 -388 CCCCCATACAACATACTTATACTCA T forward GGAGGAATGTTATTGTTTAAAGAGAT  9 -95, -89, 326 bp reverse Biotin-CAACCCCTTCTAAAAAATATCC 10 -76, -71, -69 TBX5 forward GGGTTTGGAGTTAGGTTATG 11 -645, -643,  95 bp reverse Biotin-AAATCTAAACTTACCCCCAACT 12 -628, -621 PENK forward ATATTTTATTGTATGGGTTTTTTAATAG 13 -150, -148, 322 bp reverse Biotin-ACAACCTCAACAAAAAATC 14 -139, -135,  54 bp -133, PAQR9 forward Biotin-AGATAGGGGATAATTTTAT 15 -480, -475,  54 bp reverse CCTCCCAAACTAAAATTT 16 -471, -469 LHX2 forward GTAGAAGGGAAATAAGGTTGAAA 17 +5093, 233 bp reverse Biotin-ACTAAAACCCCAATACTCCCA 18 +5102, +5113, +5125, +5127 SIM2 forward Biotin-GTGGATTTAGATTAGGATTTTGT 19 -6776, -6774, 205 bp reverse CACCCTCCCCAAATTCTT 20 -6747, -6744, -6743 ^(a)distances (nucleotides) from the transcription initiation site (+1): the positions of CpG regions on the genomic DNA used in the measurement of methylation

TABLE 3 Sequences of sequencing primers for methylation marker genes SEQ ID Gene Sequence (5′→3′) NO: CDX2 ATT AAT AGA GTT TTG TAA ATA T 21 CYP1B1 AAG GGT ATG GGA ATT G 22 VSX1 TTT GGG ATT GGG AAG 23 HOXA11 TAG TTT AGG GTA TTT TTT ATT TAT 24 T GTG AAA GTA ATG ATA TAG TAG AAA 25 TBX5 TTT GGG GGT TGG GGA 26 PENK GGG TGT TTTAGG TAG TT 27 PAQR9 CCT CCC AAA CTA AAA TTT C 28 LHX2 TGG GGG TAG AGG AGA 29 SIM2 CCT CCC CAA ATT CTT C 30

20 ng of the genomic DNA modified with bisulfite was amplified by PCR. In the PCR amplification, a PCR reaction solution (20 ng of the genomic DNA modified with bisulfite, 5 μl of 10×PCR buffer (Enzynomics, Korea), 5 units of Taq polymerase (Enzynomics, Korea), 4 μl of 2.5 mM dNTP (Solgent, Korea), and 2 μl (10 pmole/μl) of PCR primers) was used, and the PCR reaction was performed in the following conditions: predenaturation at 95° C. for 5 min, and then 45 cycles of denaturation at 95° C. for 40 sec, annealing at 60° C. for 45 sec and extension at 72° C. for 40 sec, followed by final extension at 72° C. for 5 min. The amplification of the PCR product was confirmed by electrophoresis on 2.0% agarose gel.

The amplified PCR product was treated with PyroGold reagents (Biotage, USA), and then subjected to pyrosequencing using the PSQ96MA system (Biotage, USA). After the pyrosequencing, the methylation degree of the DNA was measured by calculating the methylation index. The methylation index was calculated by determining the average rate of cytosine binding to each CpG island.

FIG. 2 quantitatively shows the methylation degree of the 10 biomarkers in the bladder cancer cell lines, measured using the pyrosequencing method. As a result, it was shown that the 10 biomarkers were all methylated at high levels in at least one of the cell lines. Table 4 below shows the promoter sequences of the 10 genes.

TABLE 4 Promoter sequences of methylation marker genes Gene SEQ ID NO: CDX2 31 CYP1B1 32 VSX1 33 HOXA11 34 T 35 TBX5 36 PENK 37 PAQR9 38 LHX2 39 SIM2 40

Example 3: Measurement of Methylation of Biomarker Genes in Urinary Cells of Bladder Cancer Patients

In order to verify whether the 10 genes can be used as biomarkers for diagnosis of bladder cancer, about 20 ml of the urine of each of 20 normal persons and 19 bladder cancer patients was centrifuged in a centrifuge (Hanil Science Industrial Co., Ltd., Korea) at 4,200×g for 10 minutes to isolate cells. The supernatant was discarded, and the cell precipitate was washed twice with 5 ml of PBS. Genomic DNA was isolated from the washed cells using the QIAamp DNA Mini kit (QIAGEN, USA), and 200 ng of the isolated genomic DNA was treated with bisulfite using the EZ DNA methylation-Gold kit (Zymo Research, USA). Then, the DNA was eluted in 20 μl of sterile distilled water and subjected to pyrosequencing.

20 ng of the genomic DNA converted with bisulfite was amplified by PCR. In the PCR amplification, a PCR reaction solution (20 ng of the genomic DNA modified with bisulfite, 5 μl of 10×PCR buffer (Enzynomics, Korea), 5 units of Taq polymerase (Enzynomics, Korea), 4 μl of 2.5 mM dNTP (Solgent, Korea), and 2 μl (10 pmole/μl) of PCR primers) was used, and the PCR reaction was performed in the following conditions: predenaturation at 95° C. for 5 min, and then 45 cycles of denaturation at 95° C. for 40 sec, annealing at 60° C. for 45 sec and extension at 72° C. for 40 sec, followed by final extension at 72° C. for 5 min. The amplification of the PCR product was confirmed by electrophoresis on 2.0% agarose gel.

The amplified PCR product was treated with PyroGold reagents (Biotage, USA), and then subjected to pyrosequencing using the PSQ96MA system (Biotage, USA). After the pyrosequencing, the methylation degree of the DNA was measured by calculating the methylation index thereof. The methylation index was calculated by determining the average rate of cytosine binding to each CpG region. After the methylation index of DNA in the urinary cells of the normal persons and the bladder cancer patients has been measured, a methylation index cut-off value for diagnosis of bladder cancer patients was determined through receiver operating characteristic (ROC) curve analysis.

FIGS. 3A-3D show measurement results for the methylation of the 10 biomarker genes in urinary cells. As can be seen, the methylation degree of the genes was higher in the sample of the bladder cancer patients than in the sample of the normal persons. Meanwhile, the methylation index in the cystitis patients and the hematuria patients was similar to that in the normal control group or was rarely higher than that in the normal control group. FIGS. 4A-4E show ROC analysis results for determining cut-off values for diagnosis of bladder cancer. Also, methylation index cut-off values for the 10 biomarkers, calculated based on the ROC curve analysis results, are shown in Table 5 below.

TABLE 5 Cut-off values for bladder cancer diagnosis of 10 biomarkers Gene cut-off (%)^(a) CDX2 5.82< CYP1B1 8.38< VSX1 29.3< HOXA11 8.81< T 11.3< TBX5 6.93< PENK 11.57< PAQR9 5.0< LHX2 13.7< SIM2 8.2<

In the analysis of the methylation of the 10 biomarkers, the methylation index of each biomarker in the clinical sample was calculated. The case in which the calculated methylation index for diagnosis of bladder cancer was higher than the cut-off value obtained through receiver operating characteristic (ROC) analysis was judged to be methylation-positive, and the case in which the calculated methylation index was lower than the cut-off value was judged to be methylation-negative.

As shown in Table 6 below and FIG. 5, when judged on the basis of the cut-off value obtained by ROC curve analysis, the urinary cells of the normal persons were methylation-negative for all the 10 biomarkers, but 12.5-62.5% of the samples of the bladder cancer patients were methylation-positive for the 10 biomarkers. Also, statistical analysis was performed and, as a result, it could be seen that 9 of the samples of the bladder cancer samples were methylation-positive for 9 of the 10 biomarkers at a significant level (p<0.01) compared to the normal person group. This suggests that 9 of the 10 methylation markers are statistically significantly methylated specifically in bladder cancer and are highly useful for diagnosing bladder cancer.

TABLE 6 Frequency of methylation-positive samples for 10 biomarkers No. of methylation-positive samples/No. of total samples (%)^(a) Gene Normal bladder cancer patient P value^(b) CDX2 0/31 (0)  9/32 (28.1) 0.002 CYP1B1 0/31 (0) 16/32 (50.0) <0.001 VSX1 0/31 (0) 14/32 (45.2) <0.001 HOXA11 0/31 (0) 17/32 (53.1) <0.001 T 0/31 (0) 15/32 (46.9) <0.001 TBX5 0/31 (0) 20/32 (62.5) <0.001 PENK 0/31 (0) 19/32 (59.4) <0.001 PAQR9 0/31 (0)  4/32 (12.5) 0.113 LHX2 0/17 (0) 13/24 (54.2) <0.001 SIM2 0/17 (0)  15/24 (62.5)0 <0.001 ^(a)frequency of methylation-positive samples; and ^(b)p values obtained through the Chi-Square test

Example 4: Evaluation of the Ability of 6 Biomarker Panel Genes to Diagnose Bladder Cancer

Using the 10 methylation biomarkers, logistic regression analysis was performed. As a result, an optimal panel of 6 genes for diagnosing bladder cancer was established. FIG. 6A shows the methylation status of the 6 biomarkers (CYP1B1, HOXA11, SIM2, PENK, LHX2 and TBX5). Whether samples were methylation-positive or methylation-negative for the 6 genes was judged according to the method described in Example 3. As a result, it could be seen that all the normal samples were methylation-negative for the 6 genes, and only the bladder cancer samples were methylation-positive for the 6 genes. Particularly, early bladder cancer samples were also methylation-positive for the 6 genes at a high frequency, suggesting that the 6 genes are highly useful for early diagnosis of bladder cancer. When the methylation of at least one gene of the gene panel consisting of the six genes was diagnosed as bladder cancer, the sensitivity and specificity of the gene panel for early bladder cancer were as extremely high as 84.0% and 100%, respectively (FIG. 6D). Also, the sensitivity and specificity of the gene panel for advanced bladder cancer were measured to be 85.7% and 100%, respectively (FIG. 6C). In addition, the sensitivity and specificity of the gene panel for all early and advanced bladder cancers were measured to be 84.4% and 100%, respectively (FIG. 6B). This suggests that the methylation of the 6 genes is highly useful for early diagnosis of bladder cancer.

Example 5: Measurement of Methylation of Biomarker Genes Using Methylated DNA-Specific Binding Protein

In order to measure the methylation of biomarkers which are methylated specifically in bladder cancer, 100 ng of the genomic DNA of each of the bladder cancer cell lines RT24 and HT1197 was sonicated (Vibra Cell, SONICS), thus obtaining about 200-400-bp genomic DNA fragments.

To obtain only methylated DNA from the genomic DNA, MBD known to bind to methylated DNA was used. Specifically, 2 μg of 6×His-tagged MBD was pre-incubated with 500 ng of the genomic DNA of E. coli JM110 (No. 2638, Biological Resource Center, Korea Research Institute of Bioscience & Biotechnology), and then bound to Ni-NTA magnetic beads (Qiagen, USA). 100 ng of the sonicated genomic DNA was allowed to react with the beads in the presence of binding buffer solution (10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 3 mM MgCl₂, 0.1% Triton-X100, 5% glycerol, 25 mg/ml BSA) at 4° C. for 20 minutes. Then, the beads were washed three times with 500 μl of a binding buffer solution containing 700 mM NaCl, and then methylated DNA bound to the MBD was isolated using the QiaQuick PCR purification kit (QIAGEN, USA).

Then, the DNA methylated DNA bound to the MBD was amplified by PCR using primers of SEQ ID NOS: 41 and 42 corresponding to the promoter region (from −6842 to −6775 bp) of the SIM2 gene.

SEQ ID NO: 41:  5′-TTC TTA TTC TCA CCA GAC ATC TCA ACA CCC-3′ SEQ ID NO: 42:  5′-ATC TCC CAT CCT CCC TCC CAC TCT C-3′

The PCR reaction was performed in the following condition: predenaturation at 94° C. for 5 min, and then 40 cycles of denaturation at 94° C. for 30 sec, annealing at 62° C. for 30 sec and extension at 72° C. for 30 sec, followed by final extension at 72° C. for 5 min. The amplification of the PCR product was confirmed by electrophoresis on 2% agarose gel.

As a result, it was seen that, for the SIM2 gene, a 168-bp amplified product was detected only in the genomic DNA of the RT24 cell line, suggesting that the gene was methylated, whereas no amplified product was detected in the HT1197 cell line, suggesting that the gene was not methylated in the HT1197 cell line (FIG. 7). Such results were consistent with the methylation measurement results obtained by the pyrosequencing method. Also, such results indicate that the use of MBD enables detection of methylated DNA.

Example 6: Evaluation of the Ability of SIM2 Gene to Diagnose Bladder Cancer by Using qMSP

In order to analyze the ability of SIM2 gene to diagnose bladder cancer, 402 sets of primers and probes, which could amplify whole CpG island of SIM2 gene and detect specific methylation sites were designed (Table 7), and methylation specific real time PCR (qMSP) was performed.

First of all, genome DNA of urine cells were isolated from urines, which were obtained from normal control 20 people and 20 bladder cancer patients respectively. Treating bisulfite to the isolated genome DNA by using EZ DNA methylation-Gold kit (Zymo Research, USA) was followed by eluting with 10 μl distilled water, and then was subjected to methylation specific real time PCR (qMSP). qMSP was performed by using bisulfite treated genome DNA as a template and methylation specific primers and probes designed according to Table 1. qMSP was performed by using Rotor-Gene Q PCR equipment (Qiagen). Total 20 μl PCR reaction solution (template DNA, 2 μl; 5× AptaTaq DNA Master (Roche Diagnostics), 4 μl; PCR primers, 2 μl (2 pmole/μl), TaqMan probe, 2 μl (2 pmole/μl); D.W. 10 μl) was prepared. Total 40 times of PCR was performed with a PCR condition that treatment at 95° C. for 5 minutes is followed by treatment at 95° C. for 15 seconds under the proper annealing temperature (58° C.˜61° C.) for 1 minute. The amplification of the PCR product was confirmed by measuring the Ct (cycling threshold) value.

Methylated and non-methylated control DNA were tested with sample DNA by using EpiTect PCR control DNA set (Qiagen, cat. no. 59695), and the sensitivity and sensitivity of set of respective primers and probes were calculated with ROC curve analysis (MedCalc Program, Belgium) (Table 8).

TABLE 7 Sequences of primer and probes for SIM2 gene qMSP Size of SEQ amplification ID Set Primer Sequences (5′→3′) product (bp) NOs: 1 F401 TTGCGTTTTTTTTC 43 44 R7 TTATTAAAAATCGC 96 Probe 7 AGATTTCGCGTAAAAGGTAGGATC 45 2 F402 TGCGTTTTTTTTCG 46 44 R7 TTATTAAAAATCGC 95 Probe 7 AGATTTCGCGTAAAAGGTAGGATC 45 3 F403 GCGTTTTTTTTCGT 47 44 R7 TTATTAAAAATCGC 94 Probe 7 AGATTTCGCGTAAAAGGTAGGATC 45 4 F404 CGTTTTTTTTCGTT 48 44 R7 TTATTAAAAATCGC 93 Probe 7 AGATTTCGCGTAAAAGGTAGGATC 45 5 F405 GTTTTTTTTCGTTT 92 49 R7 TTATTAAAAATCGC 44 Probe 7 AGATTTCGCGTAAAAGGTAGGATC 45 6 F406 TTTTTTTTCGTTTA 50 44 R7 TTATTAAAAATCGC 91 Probe 7 AGATTTCGCGTAAAAGGTAGGATC 45 7 F407 TTTTTTTCGTTTAT 51 44 R7 TTATTAAAAATCGC 90 Probe 7 AGATTTCGCGTAAAAGGTAGGATC 45 8 F408 TTTTTTCGTTTATT 52 44 R7 TTATTAAAAATCGC 89 Probe 7 AGATTTCGCGTAAAAGGTAGGATC 45 9 F409 TTTTTCGTTTATTT 53 44 R7 TTATTAAAAATCGC 88 Probe 7 AGATTTCGCGTAAAAGGTAGGATC 45 10 F410 TTTTCGTTTATTTG 54 44 R7 TTATTAAAAATCGC 87 Probe 7 AGATTTCGCGTAAAAGGTAGGATC 45 11 F411 TTTCGTTTATTTGT 55 44 R7 TTATTAAAAATCGC 86 Probe 7 AGATTTCGCGTAAAAGGTAGGATC 45 12 F412 TTCGTTTATTTGTT 56 44 R7 TTATTAAAAATCGC 85 Probe 7 AGATTTCGCGTAAAAGGTAGGATC 45 13 F413 TCGTTTATTTGTTT 57 44 R7 TTATTAAAAATCGC 84 Probe 7 AGATTTCGCGTAAAAGGTAGGATC 45 14 F414 CGTTTATTTGTTTG 58 44 R7 TTATTAAAAATCGC 83 Probe 7 AGATTTCGCGTAAAAGGTAGGATC 45 15 F415 GTTTATTTGTTTGG 59 44 R7 TTATTAAAAATCGC 82 Probe 7 AGATTTCGCGTAAAAGGTAGGATC 45 16 F416 TTTATTTGTTTGGT 60 44 R7 TTATTAAAAATCGC 81 Probe 7 AGATTTCGCGTAAAAGGTAGGATC 45 17 F417 TTATTTGTTTGGTT 61 44 R7 TTATTAAAAATCGC 80 Probe 7 AGATTTCGCGTAAAAGGTAGGATC 45 18 F418 TATTTGTTTGGTTT 62 63 R8 CTCGAAACTCTACC 140 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 19 F419 ATTTGTTTGGTTTG 65 63 R8 CTCGAAACTCTACC 139 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 20 F420 TTTGTTTGGTTTGC 66 63 R8 CTCGAAACTCTACC 138 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 21 F421 TTGTTTGGTTTGCG 67 63 R8 CTCGAAACTCTACC 137 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 22 F422 TGTTTGGTTTGCGT 68 63 R8 CTCGAAACTCTACC 136 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 23 F423 GTTTGGTTTGCGTT 69 63 R8 CTCGAAACTCTACC 135 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 24 F424 TTTGGTTTGCGTTT 70 63 R8 CTCGAAACTCTACC 134 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 25 F425 TTGGTTTGCGTTTT 71 63 R8 CTCGAAACTCTACC 133 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 26 F426 TGGTTTGCGTTTTT 72 63 R8 CTCGAAACTCTACC 132 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 27 F427 GGTTTGCGTTTTTA 73 63 R8 CTCGAAACTCTACC 131 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 28 F428 GTTTGCGTTTTTAA 74 63 R8 CTCGAAACTCTACC 130 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 29 F429 TTTGCGTTTTTAAT 75 63 R8 CTCGAAACTCTACC 129 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 30 F430 TTGCGTTTTTAATT 128 76 R8 CTCGAAACTCTACC 63 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 31 F431 TGCGTTTTTAATTA 77 63 R8 CTCGAAACTCTACC 127 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 32 F432 GCGTTTTTAATTAC 78 63 R8 CTCGAAACTCTACC 126 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 33 F433 CGTTTTTAATTACG 79 63 R8 CTCGAAACTCTACC 125 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 34 F434 GTTTTTAATTACGC 80 63 R8 CTCGAAACTCTACC 124 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 35 F435 TTTTTAATTACGCG 81 63 R8 CTCGAAACTCTACC 123 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 36 F436 TTTTAATTACGCGG 82 63 R8 CTCGAAACTCTACC 122 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 37 F437 TTTAATTACGCGGG 83 63 R8 CTCGAAACTCTACC 121 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 38 F438 TTAATTACGCGGGC 120 84 R8 CTCGAAACTCTACC 63 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 39 F439 TAATTACGCGGGCG 85 63 R8 CTCGAAACTCTACC 119 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 40 F440 AATTACGCGGGCGG 86 63 R8 CTCGAAACTCTACC 118 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 41 F441 ATTACGCGGGCGGT 87 63 R8 CTCGAAACTCTACC 117 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 42 F442 TTACGCGGGCGGTT 88 63 R8 CTCGAAACTCTACC 116 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 43 F443 TACGCGGGCGGTTT 89 63 R8 CTCGAAACTCTACC 115 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 44 F444 ACGCGGGCGGTTTC 90 63 R8 CTCGAAACTCTACC 114 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 45 F445 CGCGGGCGGTTTCG 91 63 R8 CTCGAAACTCTACC 113 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 46 F446 GCGGGCGGTTTCGA 92 63 R8 CTCGAAACTCTACC 112 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 47 F447 CGGGCGGTTTCGAG 93 63 R8 CTCGAAACTCTACC 111 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 48 F448 GGGCGGTTTCGAGA 94 63 R8 CTCGAAACTCTACC 110 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 49 F449 GGCGGTTTCGAGAT 95 63 R8 CTCGAAACTCTACC 109 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 50 F450 GCGGTTTCGAGATT 96 63 R8 CTCGAAACTCTACC 108 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 51 F451 CGGTTTCGAGATTT 97 63 R8 CTCGAAACTCTACC 107 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 52 F452 GGTTTCGAGATTTC 98 63 R8 CTCGAAACTCTACC 106 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 53 F453 GTTTCGAGATTTCG 99 63 R8 CTCGAAACTCTACC 105 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 54 F454 TTTCGAGATTTCGC 100 63 R8 CTCGAAACTCTACC 104 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 55 F455 TTCGAGATTTCGCG 103 101 R8 CTCGAAACTCTACC 63 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 56 F456 TCGAGATTTCGCGT 102 63 R8 CTCGAAACTCTACC 102 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 57 F457 CGAGATTTCGCGTA 103 63 R8 CTCGAAACTCTACC 101 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 58 F458 GAGATTTCGCGTAA 104 63 R8 CTCGAAACTCTACC 100 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 59 F459 AGATTTCGCGTAAA 105 63 R8 CTCGAAACTCTACC 99 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 60 F460 GATTTCGCGTAAAA 106 63 R8 CTCGAAACTCTACC 98 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 61 F461 ATTTCGCGTAAAAG 107 63 R8 CTCGAAACTCTACC 97 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 62 F462 TTTCGCGTAAAAGG 108 63 R8 CTCGAAACTCTACC 96 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 63 F463 TTCGCGTAAAAGGT 95 109 R8 CTCGAAACTCTACC 63 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 64 F464 TCGCGTAAAAGGTA 110 63 R8 CTCGAAACTCTACC 94 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 65 F465 CGCGTAAAAGGTAG 111 63 R8 CTCGAAACTCTACC 93 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 66 F466 GCGTAAAAGGTAGG 112 63 R8 CTCGAAACTCTACC 92 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 67 F467 CGTAAAAGGTAGGA 113 63 R8 CTCGAAACTCTACC 91 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 68 F468 GTAAAAGGTAGGAT 114 63 R8 CTCGAAACTCTACC 90 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 69 F469 TAAAAGGTAGGATC 115 63 R8 CTCGAAACTCTACC 89 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 70 F470 AAAAGGTAGGATCG 116 63 R8 CTCGAAACTCTACC 88 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 71 F471 AAAGGTAGGATCGC 117 63 R8 CTCGAAACTCTACC 87 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 72 F472 AAGGTAGGATCGCG 118 63 R8 CTCGAAACTCTACC 86 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 73 F473 AGGTAGGATCGCGA 119 63 R8 CTCGAAACTCTACC 85 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 74 F474 GGTAGGATCGCGAT 120 63 R8 CTCGAAACTCTACC 84 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 75 F475 GTAGGATCGCGATT 121 63 R8 CTCGAAACTCTACC 83 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 76 F476 TAGGATCGCGATTT 122 63 R8 CTCGAAACTCTACC 82 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 77 F477 AGGATCGCGATTTT 123 63 R8 CTCGAAACTCTACC 81 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 78 F478 GGATCGCGATTTTT 124 63 R8 CTCGAAACTCTACC 80 Probe 8 GTTGAGTCGGCGTTTAGGGTCGGG 64 79 F479 GATCGCGATTTTTA 125 126 R9 AAAACGATCACAAA 140 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 80 F480 ATCGCGATTTTTAA 139 128 R9 AAAACGATCACAAA 126 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 81 F481 TCGCGATTTTTAAT 129 126 R9 AAAACGATCACAAA 138 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 82 F482 CGCGATTTTTAATA 130 126 R9 AAAACGATCACAAA 137 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 83 F483 GCGATTTTTAATAA 131 126 R9 AAAACGATCACAAA 136 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 84 F484 CGATTTTTAATAAT 132 126 R9 AAAACGATCACAAA 135 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 85 F485 GATTTTTAATAATG 133 126 R9 AAAACGATCACAAA 134 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 86 F486 ATTTTTAATAATGA 134 126 R9 AAAACGATCACAAA 133 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 87 F487 TTTTTAATAATGAT 135 126 R9 AAAACGATCACAAA 132 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 88 F488 TTTTAATAATGATA 131 136 R9 AAAACGATCACAAA 126 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 89 F489 TTTAATAATGATAT 137 126 R9 AAAACGATCACAAA 130 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 90 F490 TTAATAATGATATT 138 126 R9 AAAACGATCACAAA 129 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 91 F491 TAATAATGATATTT 139 126 R9 AAAACGATCACAAA 128 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 92 F492 AATAATGATATTTT 140 126 R9 AAAACGATCACAAA 127 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 93 F493 ATAATGATATTTTC 141 126 R9 AAAACGATCACAAA 126 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 94 F494 TAATGATATTTTCG 142 126 R9 AAAACGATCACAAA 125 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 95 F495 AATGATATTTTCGA 143 126 R9 AAAACGATCACAAA 124 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 96 F496 ATGATATTTTCGAA 144 126 R9 AAAACGATCACAAA 123 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 97 F497 TGATATTTTCGAAA 145 126 R9 AAAACGATCACAAA 122 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 98 F498 GATATTTTCGAAAT 146 126 R9 AAAACGATCACAAA 121 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 99 F499 ATATTTTCGAAATA 147 126 R9 AAAACGATCACAAA 120 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 100 F500 TATTTTCGAAATAA 148 126 R9 AAAACGATCACAAA 119 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 101 F501 ATTTTCGAAATAAT 149 126 R9 AAAACGATCACAAA 118 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 102 F502 TTTTCGAAATAATT 150 126 R9 AAAACGATCACAAA 117 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 103 F503 TTTCGAAATAATTT 151 126 R9 AAAACGATCACAAA 116 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 104 F504 TTCGAAATAATTTT 152 126 R9 AAAACGATCACAAA 115 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 105 F505 TCGAAATAATTTTT 114 153 R9 AAAACGATCACAAA 126 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 106 F506 CGAAATAATTTTTT 154 126 R9 AAAACGATCACAAA 113 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 107 F507 GAAATAATTTTTTG 155 126 R9 AAAACGATCACAAA 112 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 108 F508 AAATAATTTTTTGT 156 126 R9 AAAACGATCACAAA 111 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 109 F509 AATAATTTTTTGTT 157 126 R9 AAAACGATCACAAA 110 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 110 F510 ATAATTTTTTGTTG 158 126 R9 AAAACGATCACAAA 109 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 111 F511 TAATTTTTTGTTGA 159 126 R9 AAAACGATCACAAA 108 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 112 F512 AATTTTTTGTTGAG 160 126 R9 AAAACGATCACAAA 107 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 113 F513 ATTTTTTGTTGAGT 161 126 R9 AAAACGATCACAAA 106 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 114 F514 TTTTTTGTTGAGTC 162 126 R9 AAAACGATCACAAA 105 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 115 F515 TTTTTGTTGAGTCG 163 126 R9 AAAACGATCACAAA 104 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 116 F516 TTTTGTTGAGTCGG 164 126 R9 AAAACGATCACAAA 103 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 117 F517 TTTGTTGAGTCGGC 165 126 R9 AAAACGATCACAAA 102 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 118 F518 TTGTTGAGTCGGCG 166 126 R9 AAAACGATCACAAA 101 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 119 F519 TGTTGAGTCGGCGT 167 126 R9 AAAACGATCACAAA 100 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 120 F520 GTTGAGTCGGCGTT 168 126 R9 AAAACGATCACAAA 99 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 121 F521 TTGAGTCGGCGTTT 169 126 R9 AAAACGATCACAAA 98 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 122 F522 TGAGTCGGCGTTTA 170 126 R9 AAAACGATCACAAA 97 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 123 F523 GAGTCGGCGTTTAG 171 126 R9 AAAACGATCACAAA 96 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 124 F524 AGTCGGCGTTTAGG 172 126 R9 AAAACGATCACAAA 95 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 125 F525 GTCGGCGTTTAGGG 173 126 R9 AAAACGATCACAAA 94 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 126 F526 TCGGCGTTTAGGGT 174 126 R9 AAAACGATCACAAA 93 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 127 F527 CGGCGTTTAGGGTC 175 126 R9 AAAACGATCACAAA 92 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 128 F528 GGCGTTTAGGGTCG 176 126 R9 AAAACGATCACAAA 91 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 129 F529 GCGTTTAGGGTCGG 177 126 R9 AAAACGATCACAAA 90 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 130 F530 CGTTTAGGGTCGGG 89 178 R9 AAAACGATCACAAA 126 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 131 F531 GTTTAGGGTCGGGG 179 126 R9 AAAACGATCACAAA 88 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 132 F532 TTTAGGGTCGGGGG 180 126 R9 AAAACGATCACAAA 87 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 133 F533 TTAGGGTCGGGGGT 181 126 R9 AAAACGATCACAAA 86 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 134 F534 TAGGGTCGGGGGTA 182 126 R9 AAAACGATCACAAA 85 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 135 F535 AGGGTCGGGGGTAG 183 126 R9 AAAACGATCACAAA 84 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 136 F536 GGGTCGGGGGTAGA 184 126 R9 AAAACGATCACAAA 83 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 137 F537 GGTCGGGGGTAGAG 185 126 R9 AAAACGATCACAAA 82 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 138 F538 GTCGGGGGTAGAGT 81 186 R9 AAAACGATCACAAA 126 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 139 F539 TCGGGGGTAGAGTT 187 126 R9 AAAACGATCACAAA 80 Probe 9 GATTTTTGGCGATCGGGGAGTTTGTT 127 140 F540 CGGGGGTAGAGTTT 188 189 R10 ACTACGAACCACAC 120 Probe 10 AGTTTTTGTCGCGTGCGTGTTCGA 190 141 F541 GGGGGTAGAGTTTC 191 189 R10 ACTACGAACCACAC 119 Probe 10 AGTTTTTGTCGCGTGCGTGTTCGA 190 142 F542 GGGGTAGAGTTTCG 192 189 R10 ACTACGAACCACAC 118 Probe 10 AGTTTTTGTCGCGTGCGTGTTCGA 190 143 F543 GGGTAGAGTTTCGA 193 189 R10 ACTACGAACCACAC 117 Probe 10 AGTTTTTGTCGCGTGCGTGTTCGA 190 144 F544 GGTAGAGTTTCGAG 194 189 R10 ACTACGAACCACAC 116 Probe 10 AGTTTTTGTCGCGTGCGTGTTCGA 190 145 F545 GTAGAGTTTCGAGT 195 189 R10 ACTACGAACCACAC 115 Probe 10 AGTTTTTGTCGCGTGCGTGTTCGA 190 146 F546 TAGAGTTTCGAGTT 196 189 R10 ACTACGAACCACAC 114 Probe 10 AGTTTTTGTCGCGTGCGTGTTCGA 190 147 F547 AGAGTTTCGAGTTT 197 189 R10 ACTACGAACCACAC 113 Probe 10 AGTTTTTGTCGCGTGCGTGTTCGA 190 148 F548 GAGTTTCGAGTTTT 198 189 R10 ACTACGAACCACAC 112 Probe 10 AGTTTTTGTCGCGTGCGTGTTCGA 190 149 F549 AGTTTCGAGTTTTT 199 189 R10 ACTACGAACCACAC 111 Probe 10 AGTTTTTGTCGCGTGCGTGTTCGA 190 150 F550 GTTTCGAGTTTTTT 200 189 R10 ACTACGAACCACAC 110 Probe 10 AGTTTTTGTCGCGTGCGTGTTCGA 190 151 F551 TTTCGAGTTTTTTT 201 189 R10 ACTACGAACCACAC 109 Probe 10 AGTTTTTGTCGCGTGCGTGTTCGA 190 152 F552 TTCGAGTTTTTTTT 202 189 R10 ACTACGAACCACAC 108 Probe 10 AGTTTTTGTCGCGTGCGTGTTCGA 190 153 F553 TCGAGTTTTTTTTG 203 189 R10 ACTACGAACCACAC 107 Probe 10 AGTTTTTGTCGCGTGCGTGTTCGA 190 154 F554 CGAGTTTTTTTTGC 204 189 R10 ACTACGAACCACAC 106 Probe 10 AGTTTTTGTCGCGTGCGTGTTCGA 190 155 F555 GAGTTTTTTTTGCG 105 205 R10 ACTACGAACCACAC 189 Probe 10 AGTTTTTGTCGCGTGCGTGTTCGA 190 156 F556 AGTTTTTTTTGCGG 206 189 R10 ACTACGAACCACAC 104 Probe 10 AGTTTTTGTCGCGTGCGTGTTCGA 190 157 F557 GTTTTTTTTGCGGA 207 189 R10 ACTACGAACCACAC 103 Probe 10 AGTTTTTGTCGCGTGCGTGTTCGA 190 158 F558 TTTTTTTTGCGGAA 208 189 R10 ACTACGAACCACAC 102 Probe 10 AGTTTTTGTCGCGTGCGTGTTCGA 190 159 F559 TTTTTTTGCGGAAT 209 189 R10 ACTACGAACCACAC 101 Probe 10 AGTTTTTGTCGCGTGCGTGTTCGA 190 160 F560 TTTTTTGCGGAATT 210 189 R10 ACTACGAACCACAC 100 Probe 10 AGTTTTTGTCGCGTGCGTGTTCGA 190 161 F561 TTTTTGCGGAATTA 211 189 R10 ACTACGAACCACAC 99 Probe 10 AGTTTTTGTCGCGTGCGTGTTCGA 190 162 F562 TTTTGCGGAATTAA 212 189 R10 ACTACGAACCACAC 98 Probe 10 AGTTTTTGTCGCGTGCGTGTTCGA 190 163 F563 TTTGCGGAATTAAG 213 189 R10 ACTACGAACCACAC 97 Probe 10 AGTTTTTGTCGCGTGCGTGTTCGA 190 164 F564 TTGCGGAATTAAGG 214 189 R10 ACTACGAACCACAC 96 Probe 10 AGTTTTTGTCGCGTGCGTGTTCGA 190 165 F565 TGCGGAATTAAGGA 215 189 R10 ACTACGAACCACAC 95 Probe 10 AGTTTTTGTCGCGTGCGTGTTCGA 190 166 F566 GCGGAATTAAGGAG 216 189 R10 ACTACGAACCACAC 94 Probe 10 AGTTTTTGTCGCGTGCGTGTTCGA 190 167 F567 CGGAATTAAGGAGA 217 189 R10 ACTACGAACCACAC 93 Probe 10 AGTTTTTGTCGCGTGCGTGTTCGA 190 168 F568 GGAATTAAGGAGAT 218 189 R10 ACTACGAACCACAC 92 Probe 10 AGTTTTTGTCGCGTGCGTGTTCGA 190 169 F569 GAATTAAGGAGATT 219 189 R10 ACTACGAACCACAC 91 Probe 10 AGTTTTTGTCGCGTGCGTGTTCGA 190 170 F570 AATTAAGGAGATTT 220 189 R10 ACTACGAACCACAC 90 Probe 10 AGTTTTTGTCGCGTGCGTGTTCGA 190 171 F571 ATTAAGGAGATTTT 221 189 R10 ACTACGAACCACAC 89 Probe 10 AGTTTTTGTCGCGTGCGTGTTCGA 190 172 F572 TTAAGGAGATTTTT 222 189 R10 ACTACGAACCACAC 88 Probe 10 AGTTTTTGTCGCGTGCGTGTTCGA 190 173 F573 TAAGGAGATTTTTG 223 189 R10 ACTACGAACCACAC 87 Probe 10 AGTTTTTGTCGCGTGCGTGTTCGA 190 174 F574 AAGGAGATTTTTGG 224 189 R10 ACTACGAACCACAC 86 Probe 10 AGTTTTTGTCGCGTGCGTGTTCGA 190 175 F575 AGGAGATTTTTGGC 225 189 R10 ACTACGAACCACAC 85 Probe 10 AGTTTTTGTCGCGTGCGTGTTCGA 190 176 F576 GGAGATTTTTGGCG 226 189 R10 ACTACGAACCACAC 84 Probe 10 AGTTTTTGTCGCGTGCGTGTTCGA 190 177 F577 GAGATTTTTGGCGA 227 189 R10 ACTACGAACCACAC 83 Probe 10 AGTTTTTGTCGCGTGCGTGTTCGA 190 178 F578 AGATTTTTGGCGAT 228 189 R10 ACTACGAACCACAC 82 Probe 10 AGTTTTTGTCGCGTGCGTGTTCGA 190 179 F579 GATTTTTGGCGATC 229 189 R10 ACTACGAACCACAC 81 Probe 10 AGTTTTTGTCGCGTGCGTGTTCGA 190 180 F580 ATTTTTGGCGATCG 80 230 R10 ACTACGAACCACAC 189 Probe 10 AGTTTTTGTCGCGTGCGTGTTCGA 190 181 F581 TTTTTGGCGATCGG 231 232 R11 GATAATAAACCCGA 140 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 182 F582 TTTTGGCGATCGGG 234 232 R11 GATAATAAACCCGA 139 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 183 F583 TTTGGCGATCGGGG 235 232 R11 GATAATAAACCCGA 138 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 184 F584 TTGGCGATCGGGGA 236 232 R11 GATAATAAACCCGA 137 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 185 F585 TGGCGATCGGGGAG 237 232 R11 GATAATAAACCCGA 136 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 186 F586 GGCGATCGGGGAGT 238 232 R11 GATAATAAACCCGA 135 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 187 F587 GCGATCGGGGAGTT 239 232 R11 GATAATAAACCCGA 134 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 188 F588 CGATCGGGGAGTTT 133 240 R11 GATAATAAACCCGA 232 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 189 F589 GATCGGGGAGTTTG 241 232 R11 GATAATAAACCCGA 132 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 190 F590 ATCGGGGAGTTTGT 242 232 R11 GATAATAAACCCGA 131 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 191 F591 TCGGGGAGTTTGTT 243 232 R11 GATAATAAACCCGA 130 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 192 F592 CGGGGAGTTTGTTT 244 232 R11 GATAATAAACCCGA 129 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 193 F593 GGGGAGTTTGTTTT 245 232 R11 GATAATAAACCCGA 128 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 194 F594 GGGAGTTTGTTTTT 246 232 R11 GATAATAAACCCGA 127 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 195 F595 GGAGTTTGTTTTTG 247 232 R11 GATAATAAACCCGA 126 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 196 F596 GAGTTTGTTTTTGT 248 232 R11 GATAATAAACCCGA 125 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 197 F597 AGTTTGTTTTTGTG 249 232 R11 GATAATAAACCCGA 124 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 198 F598 GTTTGTTTTTGTGA 250 232 R11 GATAATAAACCCGA 123 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 199 F599 TTTGTTTTTGTGAT 251 232 R11 GATAATAAACCCGA 122 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 200 F600 TTGTTTTTGTGATC 252 232 R11 GATAATAAACCCGA 121 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 201 F601 TGTTTTTGTGATCG 253 232 R11 GATAATAAACCCGA 120 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 202 F602 GTTTTTGTGATCGT 254 232 R11 GATAATAAACCCGA 119 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 203 F603 TTTTTGTGATCGTT 255 232 R11 GATAATAAACCCGA 118 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 204 F604 TTTTGTGATCGTTT 256 232 R11 GATAATAAACCCGA 117 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 205 F605 TTTGTGATCGTTTT 116 257 R11 GATAATAAACCCGA 232 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 206 F606 TTGTGATCGTTTTA 258 232 R11 GATAATAAACCCGA 115 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 207 F607 TGTGATCGTTTTAG 259 232 R11 GATAATAAACCCGA 114 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 208 F608 GTGATCGTTTTAGT 260 232 R11 GATAATAAACCCGA 113 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 209 F609 TGATCGTTTTAGTA 261 232 R11 GATAATAAACCCGA 112 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 210 F610 GATCGTTTTAGTAG 262 232 R11 GATAATAAACCCGA 111 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 211 F611 ATCGTTTTAGTAGT 263 232 R11 GATAATAAACCCGA 110 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 212 F612 TCGTTTTAGTAGTT 264 232 R11 GATAATAAACCCGA 109 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 213 F613 CGTTTTAGTAGTTT 108 265 R11 GATAATAAACCCGA 232 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 214 F614 GTTTTAGTAGTTTT 266 232 R11 GATAATAAACCCGA 107 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 215 F615 TTTTAGTAGTTTTT 267 232 R11 GATAATAAACCCGA 106 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 216 F616 TTTAGTAGTTTTTG 268 232 R11 GATAATAAACCCGA 105 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 217 F617 TTAGTAGTTTTTGT 269 232 R11 GATAATAAACCCGA 104 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 218 F618 TAGTAGTTTTTGTC 270 232 R11 GATAATAAACCCGA 103 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 219 F619 AGTAGTTTTTGTCG 271 232 R11 GATAATAAACCCGA 102 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 220 F620 GTAGTTTTTGTCGC 272 232 R11 GATAATAAACCCGA 101 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 221 F621 TAGTTTTTGTCGCG 273 232 R11 GATAATAAACCCGA 100 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 222 F622 AGTTTTTGTCGCGT 274 232 R11 GATAATAAACCCGA 99 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 223 F623 GTTTTTGTCGCGTG 275 232 R11 GATAATAAACCCGA 98 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 224 F624 TTTTTGTCGCGTGC 276 232 R11 GATAATAAACCCGA 97 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 225 F625 TTTTGTCGCGTGCG 277 232 R11 GATAATAAACCCGA 96 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 226 F626 TTTGTCGCGTGCGT 278 232 R11 GATAATAAACCCGA 95 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 227 F627 TTGTCGCGTGCGTG 279 232 R11 GATAATAAACCCGA 94 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 228 F628 TGTCGCGTGCGTGT 280 232 R11 GATAATAAACCCGA 93 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 229 F629 GTCGCGTGCGTGTT 281 232 R11 GATAATAAACCCGA 92 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 230 F630 TCGCGTGCGTGTTC 91 282 R11 GATAATAAACCCGA 232 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 231 F631 CGCGTGCGTGTTCG 283 232 R11 GATAATAAACCCGA 90 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 232 F632 GCGTGCGTGTTCGA 284 232 R11 GATAATAAACCCGA 89 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 233 F633 CGTGCGTGTTCGAG 285 232 R11 GATAATAAACCCGA 88 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 234 F634 GTGCGTGTTCGAGT 286 232 R11 GATAATAAACCCGA 87 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 235 F635 TGCGTGTTCGAGTG 287 232 R11 GATAATAAACCCGA 86 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 236 F636 GCGTGTTCGAGTGT 288 232 R11 GATAATAAACCCGA 85 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 237 F637 CGTGTTCGAGTGTG 289 232 R11 GATAATAAACCCGA 84 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 238 F638 GTGTTCGAGTGTGG 83 290 R11 GATAATAAACCCGA 232 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 239 F639 TGTTCGAGTGTGGT 291 232 R11 GATAATAAACCCGA 82 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 240 F640 GTTCGAGTGTGGTT 292 232 R11 GATAATAAACCCGA 81 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 241 F641 TTCGAGTGTGGTTC 293 232 R11 GATAATAAACCCGA 80 Probe 11 GTTTAGGGCGGGGAGAGTTGGCGAT 233 242 F642 TCGAGTGTGGTTCG 294 295 R12 CCACGCCGAACGTA 140 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 243 F643 CGAGTGTGGTTCGT 297 295 R12 CCACGCCGAACGTA 139 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 244 F644 GAGTGTGGTTCGTA 298 295 R12 CCACGCCGAACGTA 138 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 245 F645 AGTGTGGTTCGTAG 299 295 R12 CCACGCCGAACGTA 137 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 246 F646 GTGTGGTTCGTAGT 300 295 R12 CCACGCCGAACGTA 136 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 247 F647 TGTGGTTCGTAGTT 301 295 R12 CCACGCCGAACGTA 135 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 248 F648 GTGGTTCGTAGTTT 302 295 R12 CCACGCCGAACGTA 134 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 249 F649 TGGTTCGTAGTTTT 303 295 R12 CCACGCCGAACGTA 133 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 250 F650 GGTTCGTAGTTTTT 304 295 R12 CCACGCCGAACGTA 132 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 251 F651 GTTCGTAGTTTTTA 305 295 R12 CCACGCCGAACGTA 131 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 252 F652 TTCGTAGTTTTTAA 306 295 R12 CCACGCCGAACGTA 130 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 253 F653 TCGTAGTTTTTAAA 307 295 R12 CCACGCCGAACGTA 129 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 254 F654 CGTAGTTTTTAAAG 308 295 R12 CCACGCCGAACGTA 128 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 255 F655 GTAGTTTTTAAAGT 127 309 R12 CCACGCCGAACGTA 295 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 256 F656 TAGTTTTTAAAGTT 310 295 R12 CCACGCCGAACGTA 126 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 257 F657 AGTTTTTAAAGTTT 311 295 R12 CCACGCCGAACGTA 125 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 258 F658 GTTTTTAAAGTTTA 312 295 R12 CCACGCCGAACGTA 124 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 259 F659 TTTTTAAAGTTTAG 313 295 R12 CCACGCCGAACGTA 123 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 260 F660 TTTTAAAGTTTAGG 314 295 R12 CCACGCCGAACGTA 122 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 261 F661 TTTAAAGTTTAGGT 315 295 R12 CCACGCCGAACGTA 121 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 262 F662 TTAAAGTTTAGGTG 316 295 R12 CCACGCCGAACGTA 120 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 263 F663 TAAAGTTTAGGTGT 317 295 R12 CCACGCCGAACGTA 119 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 264 F664 AAAGTTTAGGTGTG 318 295 R12 CCACGCCGAACGTA 118 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 265 F665 AAGTTTAGGTGTGT 319 295 R12 CCACGCCGAACGTA 117 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 266 F666 AGTTTAGGTGTGTG 320 295 R12 CCACGCCGAACGTA 116 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 267 F667 GTTTAGGTGTGTGT 321 295 R12 CCACGCCGAACGTA 115 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 268 F668 TTTAGGTGTGTGTG 322 295 R12 CCACGCCGAACGTA 114 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 269 F669 TTAGGTGTGTGTGG 323 295 R12 CCACGCCGAACGTA 113 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 270 F670 TAGGTGTGTGTGGT 324 295 R12 CCACGCCGAACGTA 112 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 271 F671 AGGTGTGTGTGGTT 325 295 R12 CCACGCCGAACGTA 111 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 272 F672 GGTGTGTGTGGTTT 326 295 R12 CCACGCCGAACGTA 110 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 273 F673 GTGTGTGTGGTTTA 327 295 R12 CCACGCCGAACGTA 109 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 274 F674 TGTGTGTGGTTTAG 328 295 R12 CCACGCCGAACGTA 108 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 275 F675 GTGTGTGGTTTAGG 329 295 R12 CCACGCCGAACGTA 107 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 276 F676 TGTGTGGTTTAGGG 330 295 R12 CCACGCCGAACGTA 106 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 277 F677 GTGTGGTTTAGGGC 331 295 R12 CCACGCCGAACGTA 105 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 278 F678 TGTGGTTTAGGGCG 332 295 R12 CCACGCCGAACGTA 104 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 279 F679 GTGGTTTAGGGCGG 333 295 R12 CCACGCCGAACGTA 103 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 280 F680 TGGTTTAGGGCGGG 102 334 R12 CCACGCCGAACGTA 295 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 281 F681 GGTTTAGGGCGGGG 335 295 R12 CCACGCCGAACGTA 101 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 282 F682 GTTTAGGGCGGGGA 336 295 R12 CCACGCCGAACGTA 100 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 283 F683 TTTAGGGCGGGGAG 337 295 R12 CCACGCCGAACGTA 99 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 284 F684 TTAGGGCGGGGAGA 338 295 R12 CCACGCCGAACGTA 98 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 285 F685 TAGGGCGGGGAGAG 339 295 R12 CCACGCCGAACGTA 97 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 286 F686 AGGGCGGGGAGAGT 340 295 R12 CCACGCCGAACGTA 96 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 287 F687 GGGCGGGGAGAGTT 341 295 R12 CCACGCCGAACGTA 95 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 288 F688 GGCGGGGAGAGTTG 94 342 R12 CCACGCCGAACGTA 295 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 289 F689 GCGGGGAGAGTTGG 343 295 R12 CCACGCCGAACGTA 93 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 290 F690 CGGGGAGAGTTGGC 344 295 R12 CCACGCCGAACGTA 92 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 291 F691 GGGGAGAGTTGGCG 345 295 R12 CCACGCCGAACGTA 91 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 292 F692 GGGAGAGTTGGCGA 346 295 R12 CCACGCCGAACGTA 90 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 293 F693 GGAGAGTTGGCGAT 347 295 R12 CCACGCCGAACGTA 89 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 294 F694 GAGAGTTGGCGATT 348 295 R12 CCACGCCGAACGTA 88 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 295 F695 AGAGTTGGCGATTC 349 295 R12 CCACGCCGAACGTA 87 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 296 F696 GAGTTGGCGATTCG 350 295 R12 CCACGCCGAACGTA 86 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 297 F697 AGTTGGCGATTCGG 351 295 R12 CCACGCCGAACGTA 85 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 298 F698 GTTGGCGATTCGGG 352 295 R12 CCACGCCGAACGTA 84 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 299 F699 TTGGCGATTCGGGT 353 295 R12 CCACGCCGAACGTA 83 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 300 F700 TGGCGATTCGGGTT 354 295 R12 CCACGCCGAACGTA 82 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 301 F701 GGCGATTCGGGTTT 355 295 R12 CCACGCCGAACGTA 81 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 302 F702 GCGATTCGGGTTTA 356 295 R12 CCACGCCGAACGTA 80 Probe 12 GTTTGATTAGATGGGGTGCGGTTTT 296 303 F703 CGATTCGGGTTTAT 357 358 R13 TCAAAAATTCCGCC 140 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 304 F704 GATTCGGGTTTATT 360 358 R13 TCAAAAATTCCGCC 139 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 305 F705 ATTCGGGTTTATTA 138 361 R13 TCAAAAATTCCGCC 358 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 306 F706 TTCGGGTTTATTAT 362 358 R13 TCAAAAATTCCGCC 137 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 307 F707 TCGGGTTTATTATC 363 358 R13 TCAAAAATTCCGCC 136 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 308 F708 CGGGTTTATTATCG 364 358 R13 TCAAAAATTCCGCC 135 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 309 F709 GGGTTTATTATCGT 365 358 R13 TCAAAAATTCCGCC 134 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 310 F710 GGTTTATTATCGTT 366 358 R13 TCAAAAATTCCGCC 133 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 311 F711 GTTTATTATCGTTT 367 358 R13 TCAAAAATTCCGCC 132 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 312 F712 TTTATTATCGTTTT 368 358 R13 TCAAAAATTCCGCC 131 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 313 F713 TTATTATCGTTTTA 130 369 R13 TCAAAAATTCCGCC 358 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 314 F714 TATTATCGTTTTAG 370 358 R13 TCAAAAATTCCGCC 129 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 315 F715 ATTATCGTTTTAGT 371 358 R13 TCAAAAATTCCGCC 128 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 316 F716 TTATCGTTTTAGTG 372 358 R13 TCAAAAATTCCGCC 127 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 317 F717 TATCGTTTTAGTGT 373 358 R13 TCAAAAATTCCGCC 126 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 318 F718 ATCGTTTTAGTGTT 374 358 R13 TCAAAAATTCCGCC 125 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 319 F719 TCGTTTTAGTGTTA 375 358 R13 TCAAAAATTCCGCC 124 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 320 F720 CGTTTTAGTGTTAT 376 358 R13 TCAAAAATTCCGCC 123 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 321 F721 GTTTTAGTGTTATC 377 358 R13 TCAAAAATTCCGCC 122 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 322 F722 TTTTAGTGTTATCG 378 358 R13 TCAAAAATTCCGCC 121 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 323 F723 TTTAGTGTTATCGT 379 358 R13 TCAAAAATTCCGCC 120 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 324 F724 TTAGTGTTATCGTT 380 358 R13 TCAAAAATTCCGCC 119 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 325 F725 TAGTGTTATCGTTT 381 358 R13 TCAAAAATTCCGCC 118 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 326 F726 AGTGTTATCGTTTT 382 358 R13 TCAAAAATTCCGCC 117 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 327 F727 GTGTTATCGTTTTA 383 358 R13 TCAAAAATTCCGCC 116 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 328 F728 TGTTATCGTTTTAG 384 358 R13 TCAAAAATTCCGCC 115 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 329 F729 GTTATCGTTTTAGT 385 358 R13 TCAAAAATTCCGCC 114 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 330 F730 TTATCGTTTTAGTG 113 386 R13 TCAAAAATTCCGCC 358 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 331 F731 TATCGTTTTAGTGT 387 358 R13 TCAAAAATTCCGCC 112 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 332 F732 ATCGTTTTAGTGTT 388 358 R13 TCAAAAATTCCGCC 111 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 333 F733 TCGTTTTAGTGTTT 389 358 R13 TCAAAAATTCCGCC 110 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 334 F734 CGTTTTAGTGTTTG 390 358 R13 TCAAAAATTCCGCC 109 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 335 F735 GTTTTAGTGTTTGA 391 358 R13 TCAAAAATTCCGCC 108 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 336 F736 TTTTAGTGTTTGAT 392 358 R13 TCAAAAATTCCGCC 107 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 337 F737 TTTAGTGTTTGATT 393 358 R13 TCAAAAATTCCGCC 106 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 338 F738 TTAGTGTTTGATTA 105 394 R13 TCAAAAATTCCGCC 358 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 339 F739 TAGTGTTTGATTAG 395 358 R13 TCAAAAATTCCGCC 104 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 340 F740 AGTGTTTGATTAGA 396 358 R13 TCAAAAATTCCGCC 103 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 341 F741 GTGTTTGATTAGAT 397 358 R13 TCAAAAATTCCGCC 102 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 342 F742 TGTTTGATTAGATG 398 358 R13 TCAAAAATTCCGCC 101 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 343 F743 GTTTGATTAGATGG 399 358 R13 TCAAAAATTCCGCC 100 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 344 F744 TTTGATTAGATGGG 400 358 R13 TCAAAAATTCCGCC 99 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 345 F745 TTGATTAGATGGGG 401 358 R13 TCAAAAATTCCGCC 98 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 346 F746 TGATTAGATGGGGT 402 358 R13 TCAAAAATTCCGCC 97 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 347 F747 GATTAGATGGGGTG 403 358 R13 TCAAAAATTCCGCC 96 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 348 F748 ATTAGATGGGGTGC 404 358 R13 TCAAAAATTCCGCC 95 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 349 F749 TTAGATGGGGTGCG 405 358 R13 TCAAAAATTCCGCC 94 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 350 F750 TAGATGGGGTGCGG 406 358 R13 TCAAAAATTCCGCC 93 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 351 F751 AGATGGGGTGCGGT 407 358 R13 TCAAAAATTCCGCC 92 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 352 F752 GATGGGGTGCGGTT 408 358 R13 TCAAAAATTCCGCC 91 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 353 F753 ATGGGGTGCGGTTT 409 358 R13 TCAAAAATTCCGCC 90 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 354 F754 TGGGGTGCGGTTTT 410 358 R13 TCAAAAATTCCGCC 89 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 355 F755 GGGGTGCGGTTTTT 88 411 R13 TCAAAAATTCCGCC 358 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 356 F756 GGGTGCGGTTTTTA 412 358 R13 TCAAAAATTCCGCC 87 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 357 F757 GGTGCGGTTTTTAC 413 358 R13 TCAAAAATTCCGCC 86 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 358 F758 GTGCGGTTTTTACG 414 358 R13 TCAAAAATTCCGCC 85 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 359 F759 TGCGGTTTTTACGT 415 358 R13 TCAAAAATTCCGCC 84 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 360 F760 GCGGTTTTTACGTT 416 358 R13 TCAAAAATTCCGCC 83 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 361 F761 CGGTTTTTACGTTC 417 358 R13 TCAAAAATTCCGCC 82 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 362 F762 GGTTTTTACGTTCG 418 358 R13 TCAAAAATTCCGCC 81 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 363 F763 GTTTTTACGTTCGG 419 358 R13 TCAAAAATTCCGCC 80 Probe 13 AAGTTCGGTTTTCGTTCGTTTTGCGC 359 364 F764 TTTTTACGTTCGGC 420 421 R14 AAAACCCATTCATT 140 Probe 14 GTTTCGTTTTTTTTTTTTGGAAGG 422 365 F765 TTTTACGTTCGGCG 423 421 R14 AAAACCCATTCATT 139 Probe 14 GTTTCGTTTTTTTTTTTTGGAAGG 422 366 F766 TTTACGTTCGGCGT 424 421 R14 AAAACCCATTCATT 138 Probe 14 GTTTCGTTTTTTTTTTTTGGAAGG 422 367 F767 TTACGTTCGGCGTG 425 421 R14 AAAACCCATTCATT 137 Probe 14 GTTTCGTTTTTTTTTTTTGGAAGG 422 368 F768 TACGTTCGGCGTGG 426 421 R14 AAAACCCATTCATT 136 Probe 14 GTTTCGTTTTTTTTTTTTGGAAGG 422 369 F769 ACGTTCGGCGTGGT 427 421 R14 AAAACCCATTCATT 135 Probe 14 GTTTCGTTTTTTTTTTTTGGAAGG 422 370 F770 CGTTCGGCGTGGTT 428 421 R14 AAAACCCATTCATT 134 Probe 14 GTTTCGTTTTTTTTTTTTGGAAGG 422 371 F771 GTTCGGCGTGGTTT 429 421 R14 AAAACCCATTCATT 133 Probe 14 GTTTCGTTTTTTTTTTTTGGAAGG 422 372 F772 TTCGGCGTGGTTTC 430 421 R14 AAAACCCATTCATT 132 Probe 14 GTTTCGTTTTTTTTTTTTGGAAGG 422 373 F773 TCGGCGTGGTTTCG 431 421 R14 AAAACCCATTCATT 131 Probe 14 GTTTCGTTTTTTTTTTTTGGAAGG 422 374 F774 CGGCGTGGTTTCGT 432 421 R14 AAAACCCATTCATT 130 Probe 14 GTTTCGTTTTTTTTTTTTGGAAGG 422 375 F775 GGCGTGGTTTCGTC 433 421 R14 AAAACCCATTCATT 129 Probe 14 GTTTCGTTTTTTTTTTTTGGAAGG 422 376 F776 GCGTGGTTTCGTCG 434 421 R14 AAAACCCATTCATT 128 Probe 14 GTTTCGTTTTTTTTTTTTGGAAGG 422 377 F777 CGTGGTTTCGTCGT 435 421 R14 AAAACCCATTCATT 127 Probe 14 GTTTCGTTTTTTTTTTTTGGAAGG 422 378 F778 GTGGTTTCGTCGTC 436 421 R14 AAAACCCATTCATT 126 Probe 14 GTTTCGTTTTTTTTTTTTGGAAGG 422 379 F779 TGGTTTCGTCGTCG 437 421 R14 AAAACCCATTCATT 125 Probe 14 GTTTCGTTTTTTTTTTTTGGAAGG 422 380 F780 GGTTTCGTCGTCGT 124 438 R14 AAAACCCATTCATT 421 Probe 14 GTTTCGTTTTTTTTTTTTGGAAGG 422 381 F781 GTTTCGTCGTCGTT 439 421 R14 AAAACCCATTCATT 123 Probe 14 GTTTCGTTTTTTTTTTTTGGAAGG 422 382 F782 TTTCGTCGTCGTTT 440 421 R14 AAAACCCATTCATT 122 Probe 14 GTTTCGTTTTTTTTTTTTGGAAGG 422 383 F783 TTCGTCGTCGTTTA 441 421 R14 AAAACCCATTCATT 121 Probe 14 GTTTCGTTTTTTTTTTTTGGAAGG 422 384 F784 TCGTCGTCGTTTAG 442 421 R14 AAAACCCATTCATT 120 Probe 14 GTTTCGTTTTTTTTTTTTGGAAGG 422 385 F785 CGTCGTCGTTTAGA 443 421 R14 AAAACCCATTCATT 119 Probe 14 GTTTCGTTTTTTTTTTTTGGAAGG 422 386 F786 GTCGTCGTTTAGAT 444 421 R14 AAAACCCATTCATT 118 Probe 14 GTTTCGTTTTTTTTTTTTGGAAGG 422 387 F787 TCGTCGTTTAGATT 445 421 R14 AAAACCCATTCATT 117 Probe 14 GTTTCGTTTTTTTTTTTTGGAAGG 422 388 F788 CGTCGTTTAGATTT 116 446 R14 AAAACCCATTCATT 421 Probe 14 GTTTCGTTTTTTTTTTTTGGAAGG 422 389 F789 GTCGTTTAGATTTG 447 421 R14 AAAACCCATTCATT 115 Probe 14 GTTTCGTTTTTTTTTTTTGGAAGG 422 390 F790 TCGTTTAGATTTGA 448 421 R14 AAAACCCATTCATT 114 Probe 14 GTTTCGTTTTTTTTTTTTGGAAGG 422 391 F791 CGTTTAGATTTGAA 449 421 R14 AAAACCCATTCATT 113 Probe 14 GTTTCGTTTTTTTTTTTTGGAAGG 422 392 F792 GTTTAGATTTGAAG 450 421 R14 AAAACCCATTCATT 112 Probe 14 GTTTCGTTTTTTTTTTTTGGAAGG 422 393 F793 TTTAGATTTGAAGT 451 421 R14 AAAACCCATTCATT 111 Probe 14 GTTTCGTTTTTTTTTTTTGGAAGG 422 394 F794 TTAGATTTGAAGTT 452 421 R14 AAAACCCATTCATT 110 Probe 14 GTTTCGTTTTTTTTTTTTGGAAGG 422 395 F795 TAGATTTGAAGTTC 453 421 R14 AAAACCCATTCATT 109 Probe 14 GTTTCGTTTTTTTTTTTTGGAAGG 422 396 F796 AGATTTGAAGTTCG 454 421 R14 AAAACCCATTCATT 108 Probe 14 GTTTCGTTTTTTTTTTTTGGAAGG 422 397 F797 GATTTGAAGTTCGG 455 421 R14 AAAACCCATTCATT 107 Probe 14 GTTTCGTTTTTTTTTTTTGGAAGG 422 398 F798 ATTTGAAGTTCGGT 456 421 R14 AAAACCCATTCATT 106 Probe 14 GTTTCGTTTTTTTTTTTTGGAAGG 422 399 F799 TTTGAAGTTCGGTT 457 421 R14 AAAACCCATTCATT 105 Probe 14 GTTTCGTTTTTTTTTTTTGGAAGG 422 400 F800 TTGAAGTTCGGTTT 458 421 R14 AAAACCCATTCATT 104 Probe 14 GTTTCGTTTTTTTTTTTTGGAAGG 422 401 F835 TAATTAAGGAGATTTTTGGCGATC 459 460 R17 ACGAAICACACTCGAACACG 88 Probe 16 ATCGTTTTAGTAGTTTTTGTCGCGTG 461 CG

As a result of evaluating methylation of SIM2 gene using urine cell DNA from normal and bladder cancer patients, it was found that the sensitivity of SIM2 gene for bladder cancer diagnosis was 75% (15/20) 90.0% (18/20) and the high specificity of the SIM2 gene was 85% (3/20)˜95% (1/20). Such results suggest that the SIM2 methylation biomarker gene is highly useful for early diagnosis of bladder cancer.

TABLE 8 Evaluation of ability to diagnose bladder cancer using SIM2 gene Set of primers Sensitivity (%), Specificity (%), and probes Cut-off (Ct) n = 20 n = 20 1 <30.1 85 80 2 <30.0 90 80 3 <30.3 75 90 4 <30.1 85 85 5 <30.0 90 85 6 <30.5 85 90 7 <30.5 85 90 8 <30.2 90 80 9 <30.3 90 85 10 <30.5 75 95 11 <30.0 80 90 12 <30.1 80 90 13 <30.0 85 90 14 <30.2 90 90 15 <30.3 75 90 16 <30.5 80 90 17 <30.0 80 85 18 <30.3 85 90 19 <30.1 80 90 20 <30.0 75 90 21 <30.5 85 90 22 <30.5 85 90 23 <30.5 75 90 24 <30.2 75 90 25 <30.3 80 90 26 <30.5 85 85 27 <30.0 90 80 28 <30.1 85 90 29 <30.0 85 90 30 <30.1 85 80 31 <30.0 90 80 32 <30.2 90 80 33 <30.0 90 80 34 <30.1 85 85 35 <30.0 90 85 36 <30.3 90 85 37 <30.0 80 85 38 <30.5 85 85 39 <30.3 75 90 40 <30.5 85 90 41 <30.5 85 95 42 <30.0 80 90 43 <30.1 80 90 44 <30.0 85 90 45 <30.2 90 90 46 <30.3 75 95 47 <30.5 80 90 48 <30.3 85 90 49 <30.1 80 95 50 <30.0 75 90 51 <30.5 85 90 52 <30.5 85 95 53 <30.5 75 95 54 <30.2 75 90 55 <30.3 80 90 56 <30.1 85 90 57 <30.0 85 90 58 <30.5 75 95 59 <30.1 85 80 60 <30.5 75 95 61 <30.3 75 95 62 <30.1 80 95 63 <30.5 85 95 64 <30.5 75 95 65 <30.5 75 95 66 <30.3 75 90 67 <30.5 85 90 68 <30.5 85 90 69 <30.0 80 90 70 <30.1 80 90 71 <30.0 85 90 72 <30.2 90 90 73 <30.3 75 85 74 <30.5 80 85 75 <30.3 85 90 76 <30.1 80 90 77 <30.0 75 90 78 <30.5 85 95 79 <30.5 85 95 80 <30.5 75 90 81 <30.2 75 90 82 <30.3 80 95 83 <30.1 85 90 84 <30.0 85 90 85 <30.3 75 85 86 <30.5 85 90 87 <30.5 85 90 88 <30.0 80 95 89 <30.1 80 90 90 <30.0 85 90 91 <30.2 90 90 92 <30.5 80 95 93 <30.3 85 90 94 <30.0 75 90 95 <30.5 85 90 96 <30.2 75 95 97 <30.3 80 90 98 <30.1 85 90 99 <30.0 85 90 100 <30.1 85 85 101 <30.0 90 85 102 <30.3 90 85 103 <30.0 80 85 104 <30.5 85 85 105 <30.1 85 85 106 <30.0 90 85 107 <30.3 90 85 108 <30.0 80 85 109 <30.5 85 85 110 <30.0 90 80 111 <30.2 90 80 112 <30.0 90 80 113 <30.1 85 80 114 <30.0 90 80 115 <30.2 90 80 116 <30.0 90 80 117 <30.1 80 90 118 <30.0 75 90 119 <30.5 85 90 120 <30.5 85 90 121 <30.5 75 90 122 <30.2 75 90 123 <30.3 80 90 124 <30.5 85 85 125 <30.0 90 80 126 <30.1 85 90 127 <30.0 85 90 128 <30.1 85 80 129 <30.0 90 80 130 <30.2 90 80 131 <30.0 90 80 132 <30.1 85 85 133 <30.0 90 85 134 <30.3 90 85 135 <30.0 80 85 136 <30.5 85 85 137 <30.3 75 90 138 <30.5 85 90 139 <30.5 85 90 140 <30.0 80 90 141 <30.1 80 90 142 <30.0 85 90 143 <30.2 90 90 144 <30.3 75 95 145 <30.5 80 90 146 <30.3 85 90 147 <30.1 80 95 148 <30.0 75 90 149 <30.5 85 90 150 <30.5 85 95 151 <30.5 75 95 152 <30.2 75 90 153 <30.3 80 90 154 <30.1 85 90 155 <30.0 85 90 156 <30.5 75 95 157 <30.1 85 80 158 <30.5 75 95 159 <30.3 75 95 160 <30.1 80 95 161 <30.5 85 95 162 <30.5 75 95 163 <30.5 75 95 164 <30.3 75 90 165 <30.5 85 90 166 <30.5 85 90 167 <30.0 80 90 168 <30.1 80 90 169 <30.0 85 95 170 <30.2 90 95 171 <30.3 75 90 172 <30.5 80 90 173 <30.3 85 90 174 <30.1 80 90 175 <30.0 75 90 176 <30.5 85 85 177 <30.5 85 85 178 <30.5 75 90 179 <30.2 75 90 180 <30.3 80 90 181 <30.1 85 90 182 <30.0 85 95 183 <30.3 75 90 184 <30.5 85 85 185 <30.5 85 90 186 <30.0 80 90 187 <30.1 80 90 188 <30.0 85 90 189 <30.2 90 85 190 <30.5 80 90 191 <30.3 85 90 192 <30.0 75 95 193 <30.5 85 90 194 <30.2 75 90 195 <30.3 80 95 196 <30.1 85 90 197 <30.0 85 90 198 <30.1 85 85 199 <30.0 90 85 200 <30.1 85 85 201 <30.3 85 90 202 <30.1 85 85 203 <30.2 75 90 204 <30.5 85 90 205 <30.2 90 85 206 <30.5 85 90 207 <30.5 85 85 208 <30.3 80 90 209 <30.5 85 85 210 <30.0 75 90 211 <30.3 75 90 212 <30.0 80 90 213 <30.5 85 90 214 <30.5 75 95 215 <30.5 75 95 216 <30.1 85 80 217 <30.3 80 90 218 <30.5 85 90 219 <30.0 75 90 220 <30.3 75 95 221 <30.0 80 90 222 <30.5 85 90 223 <30.0 80 85 224 <30.0 90 80 225 <30.2 90 80 226 <30.1 85 90 227 <30.2 75 90 228 <30.5 75 90 229 <30.1 80 90 230 <30.1 85 80 231 <30.0 90 80 232 <30.0 80 85 233 <30.5 85 85 234 <30.0 80 85 235 <30.0 85 90 236 <30.5 85 90 237 <30.0 75 90 238 <30.0 85 90 239 <30.5 85 90 240 <30.3 75 85 241 <30.2 75 90 242 <30.0 75 90 243 <30.1 80 90 244 <30.2 90 90 245 <30.5 85 90 246 <30.5 85 90 247 <30.5 85 95 248 <30.1 85 80 249 <30.5 75 95 250 <30.2 75 90 251 <30.0 75 90 252 <30.1 80 95 253 <30.2 90 90 254 <30.5 85 95 255 <30.5 85 90 256 <30.3 90 85 257 <30.2 90 80 258 <30.0 90 80 259 <30.0 90 80 260 <30.5 75 90 261 <30.5 85 90 262 <30.3 85 90 263 <30.2 90 90 264 <30.0 85 90 265 <30.3 90 85 266 <30.0 90 85 267 <30.1 85 80 268 <30.5 85 90 269 <30.5 75 95 270 <30.3 75 90 271 <30.1 80 90 272 <30.2 75 90 273 <30.0 85 90 274 <30.0 90 80 275 <30.3 75 90 276 <30.1 80 90 277 <30.5 80 90 278 <30.1 85 90 279 <30.3 75 90 280 <30.1 80 90 281 <30.3 85 90 282 <30.3 80 95 283 <30.0 80 95 284 <30.2 90 90 285 <30.1 85 85 286 <30.2 90 80 287 <30.0 90 80 288 <30.0 75 90 289 <30.3 80 90 290 <30.1 85 85 291 <30.1 80 90 292 <30.3 85 90 293 <30.5 75 95 294 <30.0 85 90 295 <30.3 75 95 296 <30.1 80 90 297 <30.5 80 90 298 <30.5 85 85 299 <30.0 80 90 300 <30.0 85 90 301 <30.5 80 90 302 <30.0 85 90 303 <30.0 75 95 304 <30.1 80 90 305 <30.1 85 90 306 <30.2 75 90 307 <30.3 85 90 308 <30.5 85 90 309 <30.5 85 95 310 <30.5 75 95 311 <30.2 75 90 312 <30.5 85 90 313 <30.3 90 85 314 <30.0 85 90 315 <30.5 85 85 316 <30.0 90 80 317 <30.0 90 80 318 <30.3 90 85 319 <30.3 80 90 320 <30.5 85 90 321 <30.5 75 90 322 <30.5 85 95 323 <30.3 75 85 324 <30.0 85 90 325 <30.1 80 95 326 <30.5 75 95 327 <30.3 80 90 328 <30.5 75 95 329 <30.5 85 90 330 <30.3 75 95 331 <30.0 80 90 332 <30.0 80 85 333 <30.1 85 85 334 <30.1 85 90 335 <30.5 85 90 336 <30.0 80 85 337 <30.1 80 90 338 <30.3 75 90 339 <30.5 85 90 340 <30.0 90 80 341 <30.2 90 80 342 <30.0 80 90 343 <30.5 80 90 344 <30.5 85 85 345 <30.1 85 80 346 <30.5 85 85 347 <30.0 85 90 348 <30.3 85 90 349 <30.5 85 95 350 <30.0 85 90 351 <30.5 80 85 352 <30.5 85 95 353 <30.0 85 90 354 <30.1 80 90 355 <30.3 85 90 356 <30.2 75 95 357 <30.0 90 85 358 <30.1 85 85 359 <30.5 85 85 360 <30.2 90 80 361 <30.5 85 90 362 <30.1 85 80 363 <30.5 85 85 364 <30.0 85 90 365 <30.5 80 90 366 <30.5 85 95 367 <30.1 85 90 368 <30.1 80 95 369 <30.3 75 90 370 <30.2 90 95 371 <30.5 75 90 372 <30.0 85 95 373 <30.3 85 90 374 <30.0 90 85 375 <30.3 80 95 376 <30.5 80 90 377 <30.3 75 90 378 <30.1 80 90 379 <30.0 85 95 380 <30.5 75 95 381 <30.2 90 90 382 <30.3 75 90 383 <30.0 90 85 384 <30.0 90 80 385 <30.5 85 90 386 <30.0 90 85 387 <30.3 90 85 388 <30.1 85 90 389 <30.5 80 95 390 <30.1 85 90 391 <30.5 75 95 392 <30.3 75 95 393 <30.0 90 85 394 <30.0 75 90 395 <30.1 85 85 396 <30.3 80 90 397 <30.5 75 95 398 <30.0 80 90 399 <30.0 90 80 400 <30.1 80 95 401 <30.1 85 85

INDUSTRIAL APPLICABILITY

As described above in detail, the present invention provides a kit and nucleic acid chip for diagnosing bladder cancer, which can detect the methylation of CpG islands of bladder cancer-specific marker genes. It is possible to diagnose bladder cancer at an early stage of transformation using the diagnostic kit or nucleic acid chip of the present invention, thus enabling early diagnosis of bladder cancer, and the diagnostic kit or nucleic acid chip can diagnose bladder cancer in a more accurate and rapid manner compared to a conventional method.

Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only for a preferred embodiment and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof. 

What is claimed is:
 1. A method for detecting CpG methylation of SIM2 (single-minded homolog 2 (Drosophila)) gene, the method comprising the steps of: (a) isolating a genomic DNA from a clinical sample; (b) treating the genomic DNA from step (a) with bisulfite; and (c) determining hypermethylation of the CpG of the SIM2 gene in the bisulfite-treated genomic DNA from step (b) by using primer(s) to amplify a methylated CpG of the bisulfite-treated SIM2 gene.
 2. The method according to claim 1, wherein the step (c) is performed by one selected from the group consisting of PCR, methylation specific PCR, real-time methylation specific PCR, PCR using a methylated DNA-specific binding protein, quantitative PCR, pyrosequencing, and bisulfite sequencing
 3. The method according to claim 1, wherein step (c) comprises examining a CpG methylation of a promoter or exon region of SIM2 in the clinical sample.
 4. The method according to claim 3, wherein the promoter comprises a DNA sequence represented in SEQ ID NO:
 40. 5. The method according to claim 1, wherein the method further comprises the step of examining CpG methylation of a gene selected from the group consisting of TBX5—T-box 5; CDX2—caudal type homeobox transcription factor 2; CYP1B1—cytochrome P450, family 1, subfamily B, polypeptide 1; VSX1—visual system homeobox 1 homolog, CHX10-like (zebrafish); HOXA11—homeobox A11; T)—T, brachyury homolog (mouse); PENK—proenkephalin; PAQR9—progestin and adipoQ receptor family member IV; and LHX2—LIM Homeobox
 2. 6. The method according to claim 5, wherein the step of examining comprises examining CpG methylation of a promoter or exon region of the gene selected from the group consisting of TBX5; CDX2; CYP1B1; VSX1; HOXA11; T; PENK; PAQR9; and LHX2.
 7. The method according to claim 1, wherein the method further comprises the step of contacting at least one nucleic acid isolated from the clinical sample with an agent capable of determining a CpG methylation status of SIM2 gene.
 8. The method according to claim 1, wherein the primer(s) for amplifying a methylated CpG of SIM2 comprising at least one or more CpG dinucleotide in a region which hybridizes to the methylated CpG of SIM2.
 9. The method according to claim 1, wherein the primer(s) for amplifying a methylated CpG of SIM2 comprising sequence(s) having a homology of 50% or more with sequence(s) selected from the group consisting of SEQ ID NOs: 43-44, 46-63, 65-126, 128-189, 191-232, 234-295, 297-358, 360-421 and 423-460.
 10. The method according to claim 8, further comprising probe(s) capable of hybridizing with a methylated CpG of SIM2 comprising at least one or more CpG dinucleotide in a region which hybridizes to the methylated CpG of SIM2.
 11. The method according to claim 8, further comprising probe(s) capable of hybridizing with a methylated CpG of SIM2 comprising sequence(s) having a homology of 50% or more with sequence(s) selected from the group consisting of SEQ ID NOs: 45, 64, 127, 190, 233, 296, 359, 422 and
 461. 12. A method for detecting CpG methylation of SIM2—single-minded homolog 2 (Drosophila) gene for bladder carcinoma or bladder cell proliferative disorder diagnosis, the method comprising the steps of: (a) isolating a genomic DNA from a clinical sample; (b) treating the genomic DNA from step (a) with bisulfite; and (c) determining hypermethylation of the CpG of the SIM2 gene in the bisulfite-treated genomic DNA from step (b) by using primer(s) to amplify a methylated CpG of the bisulfite-treated SIM2 gene, wherein a bladder carcinoma or bladder cell proliferative disorder is detected in the human subject based on increased CpG methylation of the SIM2 gene relative to that of a control.
 13. The method according to claim 12, wherein the step (c) is performed by one selected from the group consisting of PCR, methylation specific PCR, real-time methylation specific PCR, PCR using a methylated DNA-specific binding protein, quantitative PCR, pyrosequencing, and bisulfite sequencing.
 14. The method according to claim 13, wherein the clinical sample is tissue, cell, blood, urine, serum or plasma from a patient suspected of cancer or a subject to be diagnosed.
 15. The method according to claim 12, wherein step (c) comprises examining a CpG methylation of a promoter or exon region of SIM2 in the clinical sample.
 16. The method according to claim 15, wherein the promoter comprises a DNA sequence represented in SEQ ID NO:
 40. 17. The method according to claim 12, wherein the method further comprises the step of examining CpG methylation of a gene selected from the group consisting of TBX5—T-box 5; CDX2—caudal type homeobox transcription factor 2; CYP1B1—cytochrome P450, family 1, subfamily B, polypeptide 1; VSX1—visual system homeobox 1 homolog, CHX10-like (zebrafish); HOXA11—homeobox A11; T)—T, brachyury homolog (mouse); PENK—proenkephalin; PAQR9—progestin and adipoQ receptor family member IV; and LHX2—LIM Homeobox
 2. 18. The method according to claim 17, wherein the step of examining comprises examining CpG methylation of a promoter or exon region of the gene selected from the group consisting of TBX5; CDX2; CYP1B1; VSX1; HOXA11; T; PENK; PAQR9; and LHX2.
 19. The method according to claim 12, wherein the method further comprises the step of contacting at least one nucleic acid isolated from the clinical sample with an agent capable of determining a CpG methylation status of SIM2 gene.
 20. The method according to claim 12, wherein the primer(s) for amplifying a methylated CpG of SIM2 comprising at least one or more CpG dinucleotide in a region which hybridizes to the methylated CpG of SIM2.
 21. The method according to claim 12, wherein the primer(s) for amplifying a methylated CpG of SIM2 comprising sequence(s) having a homology of 50% or more with sequence(s) selected from the group consisting of SEQ ID NOs: 43-44, 46-63, 65-126, 128-189, 191-232, 234-295, 297-358, 360-421, and 423-460.
 22. The method according to claim 20, further comprising probe(s) capable of hybridizing with a methylated CpG of SIM2 comprising at least one or more CpG dinucleotide in a region which hybridizes to the methylated CpG of SIM2.
 23. The method according to claim 20, further comprising probe(s) capable of hybridizing with a methylated CpG of SIM2 comprising sequence(s) having a homology of 50% or more with sequence(s) selected from the group consisting of SEQ ID NOs: 45, 64, 127, 190, 233, 296, 359, 422 and
 461. 